The diagnosis of periprosthetic joint infection may be difficult with patients presenting months or years after initial surgery with surgery-associated or haematogenously spread bacteria. Synovasure™ is a new point of care assay that measures alpha defensin produced by activated leucocytes in joints; it is licensed for the diagnosis of periprosthetic joint infections. We sought to include alpha defensin testing in a testing algorithm to improve the diagnosis of periprosthetic joint infection. An algorithm for testing patients with suspected periprosthetic joint infection was developed and agreed among knee surgeons in Gloucestershire, UK. Data was prospectively collected on all tests performed along with information on how the results of the alpha defensin test altered patient management. A sample of joint fluid was taken using aseptic technique in theatre and tested for alpha defensin production at the point of care. Samples were then referred for standard culture and selected samples for 16SrRNA PCR. 12 patients were assayed for alpha defensin in periprosthetic joint fluid during 2015. 7 patients were female, 5 male and ages ranged 64–86 years. 10 patients had a negative point of care alpha defensin test. Only 2 of these patients also had a leukeocyte esterase (LE) test performed and these were negative. The culture results from all samples were negative for both direct and enrichment cultures. 3 samples also had 16SrRNA PCR performed and these were negative. 2 patient samples tested positive for alpha defensin. LE tests were not performed. Both samples were culture negative on direct and enrichment culture however both samples were also referred for 16SrRNA PCR which detected DNA compatible with Staphylococcus caprae/capitis/ saccharolyticus/epidermidis from 1 patient and DNA with homology to Streptococcus gallolyticus/equinusI for the other. Alpha defensin testing improved the diagnosis of prosthetic joint infection. A positive alpha defensin test may be used to select patients for whom 16SrRNA PCR testing is useful in order to maximise the potential for pre-revision infection diagnosis and the planning of appropriate adjuncts such as antibiotic laden cement or calcium sulphate beads. Negative alpha defensin tests on aspirated joint fluid may avoid the need for arthroscopy and biopsy and allow planning for single stage revision surgery without concern for infection.