The discovery of the importance of nitric oxide to the human physiology expanded the investigation of mechanisms involved in the interactions of nitrogen oxide reactive species with biomolecules. S-nitrosothiols are a convenient source of nitric oxide for in vivo applications. Acid treatment with nitrite of compounds containing the SH group, including proteins containing cysteine residues, is a widely used method to synthesize S-nitrosothiols. In this work, several species derived from the nitrite acid treatment of human and serum albumins as well as insulin were investigated using optical absorption and fluorescence. It was demonstrated that, besides cysteine, tryptophan residues are modified and can participate in processes in vivo. Comparing the absorption spectra from human and bovine serum albumin with that from insulin, it was demonstrated that the ultraviolet absorption band, described in the literature as coming from Snitrosylation, was mainly due to N-nitrosation of tryptophan residues. Fluorescence experiments confirmed the modification of tryptophan residues, since the characteristic fluorescence peak exhibited a reduction and a blue shift. The kinetics of the new chromophores was followed by comparison of native and cysteine-blocked albumins. The kinetics of tryptophan modifications was investigated at the physiological pH using fluorescence.