Protein adsorption on orthopedic biomaterials during the initial intraoperative contact critically influences biological responses and osseointegration. Osteoconductive grafts such as β-tricalcium phosphate (β-TCP) and poly (ε-caprolactone)-β-TCP (PCL-TCP) can be functionally activated by exposure to autologous tissue. However, the composition and relevance of the resulting protein layer still remain unclear. In this study involving 10 patients undergoing primary total hip arthroplasty, β-TCP and PCL-TCP samples were incubated both in the femoral medullary cavity and within a surgical tissue collector harvesting autologous tissue (blood, bone fragments, muscle, and fat). Surface morphology was assessed microscopically, and protein adsorption was characterized via high-resolution LC-MS/MS with subsequent bioinformatics and statistical analysis. Both materials adsorbed over 2000 different autologous proteins. β-TCP showed higher overall protein concentrations, while PCL-TCP demonstrated greater proteomic diversity and incubation method-dependent shifts in protein profiles, influenced by surface roughness and wettability. Samples incubated in the tissue collector exhibited less protein variability and smaller material-specific differences compared to incubation in the femoral cavity, particularly for PCL-TCP. Predominant proteins were linked to immune regulation, stress response, and protein metabolism. These findings emphasize the impact of material properties and incubation environment on protein adsorption, with ex vivo incubation leading to more consistent protein adsorption patterns.