The finding of non-viral carriers for the delivery and release of pharmaceutical and biological compounds to ill organs and tissues is one of the most widely investigated topic in medicinal and biological chemistry in the last decades. Prior to being used as drug vehicles in the living organisms, all of the new carriers are required to be fully characterized from a physico-chemical point of view, with respect to stability, charge, size, mobility, etc. To this aim, several molecular and bulky techniques have been employed for characterization. This review considers the results obtained with a molecularly oriented spectroscopic method, i.e. electron spin resonance (ESR). The application of this technique in its various forms derived from continuous-wave (cw-ESR) and pulsed-wave (pw-ESR) modes are reviewed. In particular, carriers such as liposomes (intended for gene therapy, boron neutron capture therapy, oxymetry, and others), micelles, hydrogels, nanoparticles, dendrimers, cyclodextrins and cucurbit[n]urils are considered.