Virtually all patients with a newly discovered pleural effusion should undergo thoracentesis to aid in diagnosis and management. Pleural fluid (PF) samples should be collected into heparinized tubes to prevent clot formation. The routine PF evaluation usually includes cell count and differential, protein, lactate dehydrogenase, glucose, adenosine deaminase, cytology and, if infection is a concern, pH as well as bacterial and mycobacterial cultures. Distinguishing transudates from exudates through Lights criteria is a pragmatic first step. If the effusion is an exudate, various PF tests have proven diagnostic utility: adenosine deaminase levels > 40 U/L usually indicate tuberculosis in lymphocytic-predominant PFs or empyema in neutrophilic-predominant PFs; pH < 7.2 and glucose < 60 mg/dL allow the clinician to identify complicated parapneumonic effusions and guides their initial management; and conventional cytology may demonstrate malignant cells in 60% of the patients with malignant effusions. A number of optional PF tests may complement the diagnostic approach to an undiagnosed pleural effusion. For example, natriuretic peptide assays significantly improve the accuracy of the diagnosis of cardiac pleural effusions; PF mesothelin levels greater than 20 nM are highly suggestive of mesothelioma; and new molecular techniques have improved the identification of the causative organisms in pleural infections.