Proteome changes derived from animals that have suffered pre-slaughter stress are a fact. In this study, Proteomicanalysiswascarriedouton20bovineloinsamplesfromAsturianadelosVallesandcrossbredscattle previouslyclassifiedasnormalandDFDmeatat24hpost-mortemusingpHmeasurements.SarcoplasmicsubproteomeofLongissimusthoracisat24hpost-mortemwasfractionatedbytheuseofliquidisoelectricfocusing (OFFGEL) in the pH range 3–10, followed by SDS-PAGE analysis of each retrieved fraction. The protein fractionation profile showed high reproducibility along the different sample groups. Five protein bands showed significant differences (p < 0.05) between the two groups, allowing discrimination between them. Proteins presentinthesebands,whichwereidentifiedbyLC-MS,wereactin,phosphoglucomutase-1,alpha-crystallinB, heatshockproteinbeta-6andheatshockproteinbeta-1. Significance: The significance of this study relies on the optimization of OFFGEL fractionation as a promising technologytosearchforreliablebiomarkersofpre-slaughterstress.Thismethodseparatesproteinsalongdifferentliquidfractionsaccordingtotheirisoelectricpoint;theobtainedfractionscanbefurthercharacterizedby SDS-PAGE or directly identified by LC-MS. This achievement stands out as an alternative to the use of 2-DE electrophoresisinproteinseparationandanalysis.