Beef tenderness is a very complex and multifactorial sensorial meat quality trait, which depends partly on muscle characteristics. This tissue is very variable according to animal type (age, breed and sex) and rearing conditions. Consequently, beef tenderness exhibits a great variability. Different research programs have revealed several genes or proteins which could be good markers of beef tenderness. In order to validate the relation of these markers with beef tenderness on a large population of bovines, it is necessary to have a large-scale and trusty technique which can access different quantities of proteins related to tenderness. In this study we firstly compared Western-Blot and Dot-Blot. Secondly, we evaluated Dot-Blot technical and biological capabilities for the quantification of protein biomarkers. The results demonstrated that the Dot-Blot technique with fluorescence detection presents numerous interests. This technique allows a good reproducibility and permits the simultaneous analysis of a large number of samples. The Dot-Blot technique defined and validated in this study can be used for protein biomarkers analyses, notably to predict beef tenderness. Another major result of this study is that about 5 to 10 animals per group are required to detect large differences (> 1.5) in biomarker expression between tender and tough beef, whereas much larger numbers of animals (10 to 30) are required to detect smaller differences (about 1.2 to 1.3) taking into account the biological variability of these markers.