The determination of the protonation state of the functional groups of ligands, and the amino acid residues with electrically charged side chains (His, Lys, Arg, Asp and Glu) or the nucleotide bases of the nucleic acids that they interact with, is important for ligand binding and recognition, the enzyme activity and reaction mechanism, and protein folding/unfolding and stability. Herein, the effects of different protonation state assignments of the small substrate and inhibitors and the critical residues on the reverse transcriptase and protease of human immunodeficiency virus type 1 (HIV-1) and the M2 proton channel of influenza A virus are reviewed. Theoretical studies on these topics are summarized and compared with the experimental data.