SCOPE: The consumption of processed meat is associated with increased risk of chronic diseases, but determining how the exposure to specific cooking processes alters the metabolome is an analytical challenge. This study aims to evaluate the impact of four typical cooking methods for beef (boiling, barbecuing, grilling, and roasting) on the urinary metabolite profiles in rats, using a non-targeted approach. METHODS AND RESULTS: Male Wistar rats (n~ = ~48) are fed for 3 weeks with experimental diets containing either raw or cooked (boiled, barbecued, grilled, and roasted) beef. A control group is fed with milk proteins. The 24~h-urines are analyzed using LC-MS. The consumption of boiled meat leads to the specific excretion of di- and tri-peptides (aspartyl-leucine, glycyl-aspartate, and aspartyl-prolyl-threonine) and a cyclo-prolyl-proline (p < 0.001). No singular metabolite specifically associated with the groups "grilled," "roasted," and "barbecued" meat is observed. CONCLUSION: Urinary metabolite profiles of rats fed boiled beef are clearly distinct from those of rats fed with raw, grilled, roasted, or barbecued beef. The specific metabolites include the products of non-digested proteins and may be useful as potential intake biomarkers of this meat cooking method.