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Background: Periprosthetic shoulder infections can be difficult to diagnose. The purpose of this study was to investigate the utility of arthroscopic tissue culture for the diagnosis of infection following shoulder arthroplasty. Our hypothesis was that culture of arthroscopic biopsy tissue is a more reliable method than fluoroscopically guided shoulder aspiration for diagnosing such infection. Methods: A retrospective review identified patients who had undergone culture of arthroscopic biopsy tissue during the evaluation of a possible chronic periprosthetic shoulder infection. The culture results of the arthroscopic biopsies were compared with those of fluoroscopically guided glenohumeral aspiration and open tissue biopsy samples obtained at the time of revision surgery. Results: Nineteen patients had undergone arthroscopic biopsy to evaluate a painful shoulder arthroplasty for infection. All subsequently underwent revision surgery, and 41% of those with culture results at that time had a positive result, which included Propionibacterium acnes in each case. All arthroscopic biopsy culture results were consistent with the culture results obtained during the revision surgery, yielding 100% sensitivity, specificity, positive predictive value, and negative predictive value. In contrast, fluoroscopically guided glenohumeral aspiration yielded a sensitivity of 16.7%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 58.3%. Conclusions: Arthroscopic tissue biopsy is a reliable method for diagnosing periprosthetic shoulder infection and identifying the causative organism.