Cyclic lipopeptides (CLiPs) are amphiphilic, membrane-active, secondary metabolites produced by various organisms. They are often responsible for the plant growth-promoting and antimicrobial properties of their producers. Due to increasing resistance to pesticides and antibiotics, CLiPs have attracted more and more attention in recent years. To date, however, many CLiPs have failed on the path to drug approval or registration as biocontrol agents because their promising in vitro-activity could not be reproduced in vivo. The two main reasons for this are the lack of understanding of their mode of action and of the parameters that determine their activity and selectivity. The mode of action of CLiPs is assumed to be related to their membrane activity. Therefore, systematic structure-activity studies with model membranes are an attractive approach to gain better insight into the interactions of CLiPs with lipid membranes. A better biophysical understanding of their membrane activity could promote the rational development of highly selective CLiP-based drugs and biological control agents that are less susceptible to the emergence of resistance. The main objective of this work was to investigate the membrane interactions of CLiPs for the following: Which parameters are responsible for the membrane permeabilizing activity and selectivity of a CLiP? How do structural features of the CLiP and the membrane influence the mode of action of membrane permeabilization? In this work, three structural features were investigated for their influence on the activity and selectivity of CLiPs and on their mode of action: the acyl chain length of a CLiP, electrostatic effects between CLiP and lipid, and the thickness of the lipid membrane. To this end, the membrane permeabilization of calcein liposomes by synthetic or natural CLiPs was measured as a function of lipid concentration and lipid composition using time-correlated single-photon counting experiments. The data were then analyzed using an equi-activity analysis. This resulted partitioning isotherms that provide information about the membrane affinity of a CLiP, as well as universal dose-response curves. In contrast to typical dose-response curves, the latter represent the membrane activity of a CLiP at any lipid concentration of interest and independent of partitioning effects, in other words, the intrinsic membrane-damaging activity of a CLiP. It was found that the same structural feature may differentially affect the membrane affinity and the intrinsic membrane-damaging activity of a CLiP, with both parameters determining the membrane-permeabilizing activity and thus the selectivity of the respective CLiP. An increase in hydrophobicity or electrostatic attraction may increase the membrane affinity of a CLiP, but possibly at the expense of its intrinsic membrane-damaging activity. Moreover, the balance between these two parameters is regulated by the total lipid concentration of the system. CLiP research would benefit from an unambiguous characterization of the activity of a CLiP regardless of the experimental conditions. In this work, it is proposed to describe the activity of a CLiP by its membrane affinity and by its “molar ratio of membrane-bound CLiP per lipid to trigger 50% membrane permeabilization”. This would enable the prediction of the membrane-active CLiP concentration at any lipid concentration and could facilitate transfer and interpretation of data between different in vitro assays. In addition, electrostatic repulsion or pronounced shape and volume differences between CLiP and lipid were found to promote transient, fast membrane permeabilization, presumably by an asymmetry stress-induced mode of action. Furthermore, it was shown that the intrinsic membrane-damaging activity of a clip decreases with increasing membrane thickness. This is explained by a higher membrane stability of thicker membranes. Knowledge of the position of a CLiP in the membrane is critical for understanding its mode of action at the molecular level. Therefore, another aim was to determine the position and orientation of a CLiP in a model membrane. To this end, the sensitivity of tryptophan fluorescence to its environment was exploited and the intrinsic fluorescence properties of four tryptophan-containing CLiP variants was investigated by time-resolved emission spectra and anisotropy, fluorescence quenching, and total intensity changes. Together, the data describe the immediate environment of a CLiP, its membrane affinity and mobility within the membrane, and membrane order. Preliminary results suggest that this approach, in combination with MD simulations, may provide a sophisticated analysis tool for determining CLiP position and orientation in the lipid membrane. Further experiments are required to confirm this hypothesis. In conclusion, this work has contributed to a better biophysical understanding of the membrane interactions of CLiPs responsible for membrane permeabilization.