Recent studies have indicated that tribochemical reaction layers form on metal-on-metal-bearing surfaces, which may play a significant role in the performance and longevity of artificial joints. The purpose of this study was to determine the role of friction in the formation of tribofilms, and an in situ atomic force microscopy single-asperity sliding setup was used to perform in situ microscopic friction experiments to control the contact area and load. Time-of-flight secondary ion mass spectrometry and Raman spectroscopy were also employed to investigate changes in the composition and structure of the proteins at different sliding cycles. The results revealed that the proteins first unfolded under shear and then underwent chain breakage, dehydrogenation, and desulfurization over time as friction progressed. Finally, the carbonaceous fragments did not show graphitization trends under only shear stress.