Periprosthetic joint infection (PJI) is a serious complication that may occur after native joint replacement leading to a severe health and economic burden. Currently, due to several confounding factors, PJI is difficult to diagnose. Today, a multidisciplinary approach is indispensable to correctly define a periprosthetic joint infection; indeed, tissue histology, microbiology cultures and clinical findings are used together to achieve this goal. Analysis of α-defensin is commonly used for PJI diagnosis, as it allows the rapid detection of α-defensin present in the synovial fluid following a microbial infection. Currently, a point-of-care testing (POCT) assay able to detect the presence of human α-defensins 1-3 in synovial fluid of patients is aimed directly at orthopedic surgeons. However, many orthopedic surgeons lack experience and training in quality laboratory practices, often failing to appreciate the significance of quality control and proper documentation when using POCT assays. To guarantee the highest quality diagnostic services, the α-defensin test should be used together with other biochemical and microbiological criteria commonly used for PJI diagnosis. Additionally, the close cooperation and communication between laboratory, pathologists and physicians is of fundamental importance in the correct diagnosis of PJI.