Aims. The diagnosis of periprosthetic joint infection (PJI) remains a challenge, as no single diagnostic test shows high diagnostic accuracy. Recently, the measurement of synovial biomarkers has shown promising results. The aim of this study was to present a novel multiplex micro-enzyme-linked immunosorbent assay (ELISA) method for the rapid and simultaneous measurement of alpha-defensin, interleukin (IL)-6, and calprotectin developed in a model buffer system and human spiked synovial fluid. Methods. A microfluidics- and chemiluminescence-based multiplex micro-ELISA point-of-care testing system was employed for the rapid and simultaneous measurement of alpha-defensin, calprotectin, and IL-6 developed in a model buffer system and spiked human synovial fluid. Cut-off values of 1.56 µg/ml for alpha-defensin, 50 µg/ml for calprotectin, and 0.031 µg/ml for IL-6 were extracted from the literature as optimal cut-off values for the diagnosis of PJI, and were used for comparison. Results. Limit of detection (LoD) was determined for each individual biomarker by means of calibration curves with serial dilutions in a model buffer system. LoDs of 0.008, 0.002, and 0.00014 µg/ml were determined for alpha-defensin, calprotectin, and IL-6, respectively. The spiked synovial fluid assay determined LoDs of 0.08, 0.31, and 0.004 µg/ml for alpha-defensin, calprotectin, and IL-6, respectively. Conclusion. These findings highlight the proposed platform’s unique features, including simultaneous measurement of three key synovial biomarkers, minimal sample volume requirements (5 to 50 µl), lower LoDs compared to conventional tests, and a short processing turnaround time of 22 minutes. Further validation studies are necessary to confirm its clinical utility. Cite this article: Bone Joint Res 2025;14(3):176–184