The elucidation of the subcellular distribution of proteins under different conditions is a major challenge in cell biology. This challenge is further complicated by the multicompartmental and dynamic nature of protein localization. To address this issue, quantitative proteomics workflows have been developed to reliably identify the protein complement of whole organelles, as well as for protein assignment to subcellular location and relative protein quantification based on different cell culture conditions. Here, we review quantitative MS‐based approaches that combine cellular fractionation with proteomic analysis. The application of these methods to the characterization of organellar composition and to the determination of the dynamic nature of protein complexes is improving our understanding of protein functions and dynamics.