ABSTRACT Background: Pleural effusion is a collection of fluid in the Pleural space. Determining the cause of a pleural effusion is greatly facilitated by analysis of the pleural fluid. Etiological diagnosis in of those cases is a diagnostic challenge to the clinician because even after all cumbersome work-ups some cases remain undiagnosed. Pleural effusion remains undiagnosed after routine tests in pleural fluid in many patients. so, we need a simple and safe investigative tool to evaluate undiagnosed effusion. This study is designed to diagnose the cases of undiagnosed effusions by a simple and safe investigative tool. Materials & Methods: A hospital based prospective study done in 30 patients with exudative pleural effusion remain undiagnosed after pleural fluid analysis in department of respiratory medicine at government medical college, Bhilwara, Rajasthan during one-year period. Pleural fluid sent for basic biochemical and microbiological investigations. Pleural biopsy was performed using ABRAM’S pleural biopsy needle under strict aseptic precaution under local anesthesia and the specimen was sent for the following investigations such as histopathological examination, rt-PCR and culture for Tuberculosis by BACTEC. Results: Histopathological examination diagnosed 46.66% of biopsy specimens as tuberculosis and 23.33% as malignancy. The rest 30% of case were left undiagnosed after histopathological examination of Pleural biopsy. Reverse Transcriptase Polymerase Chain reaction was positive in16.66% of the cases and was negative in 83.33% of the cases. Our study shows that tuberculous pleural effusion is more common in the younger age group and malignant pleural effusion more common in the elder age in both males and females. There were no false positive results with histopathological examination, BACTEC & rt PCR making the specificity and positive predictive value as 100%. The sensitivity was 70%, 10% & 25% respectively and negative predictive value was60%, 35% & 40% respectively in tuberculosis patients. Conclusion: We concluded that combination of histopathological examination, BACTEC culture and rtPCR reaction in closed pleural biopsy has a greater diagnostic yield in diagnosing exudative effusions not diagnosed by pleural fluid analysis. Closed pleural biopsy can be used as a easy, quick, cost effective and relatively safe method.