Even though analysis of peritoneal fluids (PF) is often requested to medical laboratories for biochemical and morphological tests, there is still no mutual agreement on what the most appropriate way is to manage PF samples and which tests should be appropriately executed. In this update, we tried to identify the most useful tests for PF analysis to establish best practice indications. We performed a literature review and examined available guidelines to select the most appropriate tests by an evidence-based approach. Accordingly, the basic PF profile should include (1) serum to effusion albumin gradient and (2) automated cell counts with differential analysis. This profile allows to determine the PF nature, differentiating between ‘high-albumin gradient’ and ‘low-albumin gradient’ effusions, which helps to identify the pathophysiological process causing the ascites formation. Restricted to specific clinical situations, additional tests can be requested as follows: PF lactate dehydrogenase (LDH) and glucose, to exclude (LDH) or confirm (glucose) secondary bacterial peritonitis; PF total protein, to differentiate ascites of cardiac origin from other causes; PF (pancreatic) amylase, for the identification of pancreatic ascites; PF bilirubin, when a choleperitoneum is suspected; PF triglycerides, in differentiating chylous from pseudochylous ascites and PF creatinine, to detect intraperitoneal urinary leakage.