Proteomics constitutes a useful tool for the study of protein from cells or tissues. The main techniques are based on the separation of proteins by electrophoresis or chromatography and their identification by mass spectrometry. This approach, combined with the use of bioinformatics tools, allows the construction of functional interactomes. During the last few years, proteomics was in a phase of expansion in medicine, pharmacology, and agronomy. In this article, we will focus on proteomics as applied to beef production. Protein maps of cattle skeletal muscle have been constructed, and comparative proteomics has provided new insights into the knowledge of muscle development and its regulation. New pathways involved in the origin of muscle hypertrophy have been identified. Proteomics also allowed to providing a list of markers of beef tenderness, which will be used in the conception of immunological tools such as antibody microarray, applied in the evaluation of the tenderness potential of cattle. The use of bioinformatics tools allowed a better knowledge of the functional mechanisms at the origin of beef tenderness.