Exploring the tendon proteome is a challenging but important task for understanding the mechanisms of physiological/pathological processes during ageing and disease and for the development of new treatments. Several extraction methods have been utilised for tendon mass spectrometry, however different extraction methods have not been simultaneously compared. In the present study we compared protein extraction in tendon with two chaotropic agents, guanidine hydrochloride (GnHCl) and urea, a detergent, RapiGest™, and their combinations for shotgun mass spectrometry. An initial proteomic analysis was performed following urea, GnHCl, and RapiGest™ extraction of equine superficial digital flexor tendon (SDFT) tissue. Subsequently, another proteomic analysis was performed following extraction with GnHCl, Rapigest™, and their combinations. Between the two chaotropic agents, GnHCl extracted more proteins, whilst a greater number of proteins were solely identified after Rapigest™ extraction. Protein extraction with a combination of GnHCl followed by RapiGest™ on the insoluble pellet demonstrated, after label‐free quantification, increased abundance of identified collagen proteins and low sample to sample variability. In contrast, GnHCl extraction on its own showed increased abundance of identified proteoglycans and cellular proteins. Therefore, the selection of protein extraction method for tendon tissue for mass spectrometry analysis should reflect the focus of the study.