Fluoroquinolones are widely employed to treat or prevent respiratory diseases in poultry, and their residues could be present even after slaughtering. Thus, this study aimed to assess the oxidative effect of fluoroquinolones on myosin and chicken meat proteins under in vitro conditions, using proteomics and peptidomics approaches. Myosin was initially exposed to ciprofloxacin and sarafloxacin, which showed an increase in protein carbonylation over time, especially with sarafloxacin. Then, chicken meat was skipped with sarafloxacin to assess its oxidative effect on in vitro gastrointestinal digestion, and the main proteins were isolated and subsequently identified by mass spectrometry in tandem. The proteins more susceptible to oxidation by sarafloxacin were myosin, β-enolase, CK-M-type, and actin. After peptidomics analysis, collagen peptides resulted as most prone to oxidative damage. These findings confirm the potential negative effects of sarafloxacin residues on chicken meat which could affect its nutritional and technological properties.