The objective of this study was to validate the Enhanced Peritoneal Stripe Sign (EPSS) in diagnosing pneumoperitoneum in patients presenting with acute abdomen. The EPSS was described as a specific sonographic sign of pneumoperitoneum in an animal model and few patients who had undergone laparoscopy (Muradali et al. in Am J Roentgenol 173(5): 1257-1262, 1999). This is the first large-scale study in patients to detect the efficacy of EPSS. Six hundred consecutive patients with acute abdominal pain presenting to the author over a period of 3 months in the emergency ultrasonography department were prospectively studied for the presence of the EPSS. As part of their clinical work up, patients also underwent plain radiographs and/or a computed tomography (CT) of the abdomen. The author was unaware of the results of other imaging studies at the time of the sonographic examination. In all cases, the final diagnosis was based on the intra-operative findings, results of other imaging techniques and clinical follow-up. Based on the final diagnosis, 21 out of 600 patients had pneumoperitoneum. The EPSS was found to be positive in all 21 of these patients. Another three patients were found to have the sign false positive. There were no false negatives in this study. The EPSS thus had a sensitivity of 100%, a specificity of 99%, a positive predictive value of 87.5% and a negative predictive value of 100%. The EPSS is a reliable and accurate sonographic sign for the diagnosis of pneumoperitoneum. It should be looked for in all patients presenting with acute abdominal pain.