Peptides, as part of the beyond the rule of 5 (bRo5) chemical space, represent a unique class of pharmaceutical compounds. Because of their exceptional position in the chemical space between traditional small molecules (molecular weight (MW) < 500 Da) and large therapeutic proteins (MW > 5000 Da), peptides became promising candidates for targeting challenging binding sites, including even targets traditionally considered as undruggable – e.g. intracellular protein–protein interactions. However, basic knowledge about physicochemical properties that are important for a drug to be membrane permeable is missing but would enhance the drug discovery process of bRo5 molecules. Consequently, there is a demand for quick and simple lipophilicity determination methods for peptides. In comparison to the traditional lipophilicity determination methods via shake flask and in silico prediction, chromatography-based methods could have multiple benefits such as the requirement of low analyte amount, insensitivity to impurities and high throughput. Herein we elucidate the role of peptide lipophilicity and different lipophilicity values. Further, we summarize peptide analysis via common chromatographic techniques, in specific reversed phase liquid chromatography, hydrophilic interaction liquid chromatography and supercritical fluid chromatography and their role in drug discovery and development process.