Background <p>Accurate and timely diagnosis of periprosthetic joint infection (PJI) is of utmost importance. Although synovial alpha-defensin has shown potential as a biomarker, recent studies have questioned its additional benefit over traditional synovial biomarkers and advised against its routine use. The primary objective is to evaluate the diagnostic accuracy of the alpha-defensin immunoassay in PJI. Secondary objectives include comparing its diagnostic accuracy against traditional biomarkers and assessing our results in the context of existing research to provide a solid perspective on its clinical utility.</p> Methods <p>This is a prospective cohort study. Synovial samples were obtained at the time of revision arthroplasty or from painful arthroplasties. A complete laboratory workup was performed, including CBC, ESR, CRP, WBCs count. Synovial samples were analyzed for leucocytic count, PMN percentage, leucocyte esterase, and alpha-defensin immunoassay. Culture and sensitivity, and histopathology were also done. Patients who met the inclusion criteria were classified into septic and aseptic according to MSIS criteria.</p> Results <p>Ninety joints met our inclusion criteria. Alpha-defensin immunoassay was positive in 36 joints and negative in 54 joints, with 1 false positive and 3 false negatives, resulting in a sensitivity of 92.11% (95% CI, 78.62–98.34%), a specificity of 98.08% (95% CI, 89.74–99.95%), positive predictive value (PPV) of 49.43% (95% CI, 12.28–87.22%), negative predictive value (NPV) of 99.84% (95% CI, 99.52–99.94%) and diagnostic accuracy of 97.96% (95% CI, 92.48–99.78%). The optimal cutoff was 9.2, and the area under the curve (AUC) was 0.945.</p> Conclusions <p>While the alpha defensin immunoassay is not recommended to be used routinely as a screening method for PJI, its high specificity and NPV make it a valuable addition to traditional blood and synovial parameters in the diagnosis of complex hip and knee PJI, particularly for ruling out infection.</p>