Tendons play a fundamental role in the musculoskeletal system and locomotion by transferring forces generated from muscles to the skeleton. Tendon injuries can occur due to sports related incidents, as a result of trauma to overuse or during disease or ageing. Using proteomic techniques tendon protein profiles could be defined under different conditions. The aim of this study was to optimise tendon sample preparation for mass spectrometry analysis, which will be valuable for future tendon studies.