<p>Copper complexes of 1,10-phenanthroline (phen) and related α-diimines present high potential as antitumor agents. Despite this, their selectivity toward cancer cells still needs improvement, and establishing clear structure–activity relationships for rational design remains elusive. To identify chemical factors that may underlie their biological behavior but remain poorly understood, we investigated the speciation equilibria of Cu-α-diimine-dipeptide systems by potentiometry and assessed how different factors influence their cytotoxic activity in human ovarian cancer cells (A2780 and A2780-cisR). The results indicate that both homoleptic Cu-α-diimine and heteroleptic Cu-α-diimine-dipeptide species are relevant even at the micromolar concentrations used in biological assays. Copper enhances the cytotoxicity of α-diimines in the order neo (2,9-dimethyl-1,10-phenanthroline) &gt; phen &gt; tmp (3,4,7,8-tetramethyl-1,10-phenanthroline), through the formation of complexes expected to predominate in the culture medium and by promoting cellular uptake. The copper-bound α-diimine in Cu-α-diimine-dipeptide systems proved to be a key factor in cytotoxicity, whereas the coordinated dipeptide modulates it only at short incubation times. Dipeptides can be considered models of biological ligands that interact with Cu-α-diimine species. Indeed, both dipeptides and albumin have minimal effects on the cytotoxicity of Cu-α-diimine systems. The mixed-ligand Cu-α-diimine-dipeptide complexes with neo are the most stable and predominate under simulated biological conditions. Thus, compared with phen and tmp, neo is expected to remain coordinated to copper in biological media, further enhancing its cytotoxicity.</p>