Investigation of the triazole-derived drugs action mechanisms and understanding of their affinity and specificity molecular basis may contribute to the new drugs development. The study was aimed to investigate the triazoles class representative (fluconazole) complexes with amino acids using mass spectrometry, molecular dynamics and ab initio quantum chemistry calculations. During the experimental study, the fluconazole, alanine, lysine and threonine solutions were analyzed by electrospray ionization mass spectrometry and tandem mass spectrometry. The molecular dynamics modeling of the fluconazole–amino acid complexes was performed using the CHARMM force field. The quantum chemistry calculations of the complexes structure and energy parameters were carried out using the density-functional theory by B3LYP calculations (3-21G and 6-311++G** basis sets). Mass spectra indicated that fluconazole formed stable complexes with amino acids in the 1 : 1 stoichiometric ratio. In accordance with the tandem mass spectrometry with varying fluconazole–amino acid associates ion fragmentation energy, the following sequence was obtained: [Fluc + Ala + H]+ < [Fluc + Lys + H]+ < [Fluc + Thr + H]+. The fluconazole–amino acid interaction energy values resulting from the quantum chemistry calculations formed the sequence similar to that obtained by experiment. Thus, as seen in the case of fluconazole–amino acid complexes, it is possible to combine the experimental mass spectrometry studies with quantum chemical modeling for the complexes properties assessment.