The present study investigated chemically modified gelatin biopolymer films. Gelatin solutions were treated with glyoxal and glycolaldehyde, respectively, at concentrations ranging from 0.25 to 7.5 wt % based on gelatin. From these solutions, films were produced under defined conditions and characterized with different chemical and physical methods. N(epsilon)-carboxymethyllysine (CML), glyoxal-derived lysine dimer (GOLD), and 5-(2-imino-5-oxo-1-imidazolidinyl)norvaline (imidazolinone) were analyzed as chemical parameters for protein modification by reversed-phase high-performance liquid chromatography (RP-HPLC) and fluorescence detection after post-column o-phthaldialdehyde derivatization. An increase in the content of these substances with increasing concentrations of carbonyl modifiers correlated with the loss of available free lysine and arginine residues. Swelling, solubility, and mechanical properties (Young's modulus, stress and strain at break) showed the relationship with the degree of monovalent modification and cross-linking as well. The determination of unreacted glyoxal and glycolaldehyde suggested a different mechanism of cross-linking induced by glyoxal versus glycolaldehyde as reactive intermediates in Maillard chemistry.