The Eph receptor-ephrin system is an emerging target for the development of novel antiangiogenetic agents. We recently identified lithocholic acid (LCA) as a small molecule able to block EphA2-dependent signals in cancer cells, suggesting that its (5β)-cholan-24-oic acid scaffold can be used as a template to design a new generation of improved EphA2 antagonists. Here, we report the design and synthesis of an extended set of LCA derivatives obtained by conjugation of its carboxyl group with different α-amino acids. Structure-activity relationships indicate that the presence of a lipophilic amino acid side chain is fundamental to achieve good potencies. The l-Trp derivative (20, PCM126) was the most potent antagonist of the series disrupting EphA2-ephrinA1 interaction and blocking EphA2 phosphorylation in prostate cancer cells at low μM concentrations, thus being significantly more potent than LCA. Compound 20 is among the most potent small-molecule antagonists of the EphA2 receptor.