Reverse Phase Protein Arrays (RPPA) were applied for the quantification and validation of protein biomarkers of beef qualities on M. longissimus thoracis sampled early post-mortem from young Charolais bulls. pHu was related to six proteins, three of which are glycolytic enzymes (ENO1, ENO3 and TPI1), while others belong to structural (TTN and α-actin) and proteolytic (μ-calpain) pathways. For color traits, several correlations were found, interestingly with structural proteins. The relationships were in some cases trait-dependent. To understand the mechanisms and explore animal variability, color data were categorized into three classes. α-actin and TTN allowed efficient separation of the classes and were strongly related with all color traits. Biomarkers belonging to heat stress and metabolism pathways were also involved. Two identified proteins, namely Four and a half LIM domains 1 (FHL1) and Tripartite motif-containing 72 (TRIM72), were for the first time related to beef color. Overall, these relationships could be used to develop muscle-specific processing strategies to improve beef color stability.