Abstract Background: The current diagnostic cornerstone for septic arthritis contains gram stains, bacterial culture, and cell count with a differential of aspirated synovial fluid. Recently, a synovial leukocyte esterase (LE) test has been used for diagnosing septic arthritis. Since this test measures the esterase activity of leukocytes, there is always a dilemma for using this test in patients with inflammatory arthritis. Methods: We collected the synovial fluid specimens as part of the general diagnostic protocol for patients suspected of Juvenile Idiopathic Arthritis (JIA) or Septic Arthritis (SA). Each group included 34 patients. We compared the result of the synovial LE test with the result of the culture of each patient. Results: The mean ages of patients were 64.14 ± 31.27 and 50.88 ± 23.19 months in the JIA group and septic arthritis group, respectively. The LE test results were positive in 30 specimens, trace in 3 and negative in one in the first-time test and were positive in 31 specimens and trace in 3 in the second-time test, while it was negative in all patients with JIA. Hence, the sensitivity of the synovial LE test was 80.8%, the specificity, PPV, and NPV were 78.6, 70.0, 86.8% respectively based on a positive culture. Conclusion: The leukocyte esterase strip test can be used as a rapid, bedside method for diagnosing or excluding bacterial infections in different body fluids. The synovial LE test can be used as an accurate test to rapidly rule in or out an acute articular bacterial infection, even in patients with concurrent inflammatory arthritis.