Since no biological activity has been reported for Sanguinamide A, we turned to a target agnostic cell painting assay to see whether morphological changes could be observed upon treatment. Indeed, Sanguinamide A induced significant changes in U2OS cells in comparison to a DMSO control allowing comparison with the other compounds. Although such changes can be induced by via extracellular receptor binding, we believe that they are caused by interaction with intracellular targets as the induction values correlated well with the CHI-IAM and Caco-2 values. Even compound SA-B2 was observed to elicit morphological changes at the highest tested concentration (50 μM) even though its permeability was mediocre in the Caco-2 assay. All the peptides with low permeability also did not induce any changes in the cell painting parameters. Interestingly, the pyridine containing SA-B2 was the only compound that could be considered similar to the parent compound Sanguinamide A. All other modifications, including the tert-butyl glycine in Danamide F, led to compounds with more significantly changed profiles. Similarly, when compared to Danamide F, only the benzene containing SA-B3 was found to reach the similarity threshold. These results might be driven by the local polarity of the backbone aromatic group, although the cLogP values of these compounds do not explain these observations as they consider the entire peptide. Nonetheless, it is clear that very subtle single atom changes lead to compounds with different biological activity profiles even when their membrane permeability is similar.