Although the impact of oxidation on human health has been of growing interest, the oxidation of proteins, major component of meat, has received little attention. This paper describes the in vitro effect of five fluoroquinolones (FQs) on carbonylation of sarcoplasmic and myofibrillar proteins of beef when found at concentrations close to the maximum residue limit (MRL). Samples were treated individually with the FQs, determining in each protein fraction the carbonyl index, protein content and oxidized proteins identification, using 2,4-dinitrophenyhydrazine (DNPH) alkaline assay, Western blot and Bradford methods, and mass spectrometry, respectively. Besides, the in vitro effect of these residues on gastric and duodenal digestion of proteins was evaluated. The carbonylation induced by FQs affected both protein fractions being significant with respect to the blank in 73.3% of cases. This damage was correlated with loss of solubility and digestibility, with sarcoplasmic proteins the most affected. Danofloxacin and enrofloxacin were the FQs with greatest oxidant effects, especially affecting glycolysis and glycogen proteins. Our results suggest that these residues induce irreversible oxidative damage on the main beef proteins and could affect their nutritional value.