The stability constants, enthalpy ΔH 0, entropy ΔS 0, and Gibbs energy ΔG 0 were determined for the host–guest complexes (1:1) of calix[4]arene bis-hydroxymethylphosphous acid with glycine, l-alanine, l-valine, l-leucine, l-isoleucine residues in methanol solution with the aid of the titration experiments followed by calorimetric and spectroscopic (1H NMR, UV) methods. The experimental data indicated that the host–guest complexation was under control of the direct electrostatic interaction between negatively charged calixarene phosphoryl group and amino acid residue NH 3 + group, modulated by the hydrophobic interaction, which drive the inclusion of the residue alkyl side-chain into the calixarene cavity. The stability of the inclusion complexes was found correlated with the size of the aliphatic amino acid’s side-chain. The experimental data were additionally analyzed in the terms of the three state model corresponding to coexistence of 2:1 and 1:1 complexation equilibria.