Ultra-high molecular-weight polyethylene (UHMWPE) has been used in total joint replacement for the last three decades. Despite the recent advancements in prosthesis design, the wear of UHMWPE remains a serious clinical problem; the release of wear debris may induce osteolysis and implant loosening. Controlling the quality of the polyethylene is essential to improve its wear resistance and any potential adverse effect caused by processing, manufacturing or sterilisation should be avoided. To evaluate the influence of the sterilisation method (gamma-irradiation and ethylene oxide (EtO)-treatment) and third-body particles, gamma- and EtO-sterilised UHMWPE acetabular cups were tested against CoCrMo femoral heads in a hip joint simulator run for 2.5 million cycles in bovine calf serum in the presence of third-body polymethylmethacrylate (PMMA) particles. A method not requiring ultra-centrifugation has been proposed for the isolation of polyethylene wear debris from the serum lubricant. SEM analysis allowed debris shape and morphology to be determined, and the wear mechanism operating in this study to be hypothesised. The morphological features of the wear debris were in agreement with clinical findings, enabling the hip simulator function to be validated. Micro-Raman spectroscopy coupled to PLS analysis showed that the mechanical friction during in vitro tests induced significant crystallinity changes in all the cups. The most significant changes were observed for the EtO-sterilised cups, which showed the highest weight loss.