Objective: Pleural effusion may occur in patients due to benign or malignant processes. This study aimed to evaluate the efficacy and safety of Uniportal Video-Assisted Thoracoscopic Surgery (VATS) operation in the diagnosis processes of patients with pleural effusion.Methods: The data of 50 patients who underwent diagnostic Uniportal VATS pleural biopsy between November 2018 and April 2021 were retrospectively analyzed. The patients' age, gender, postoperative hospital stay, operation-related complications, and pathology results of the pleural tissues were recorded.Results: Thirty-five (70%) cases were male, 15 (30%) were female, mean age was 52.58 ± 20.1, mean operative time was 56.1 ± 16.8 minutes, and mean postoperative hospital stay was 2.64 ± 0.89 days. 48 (96%) of the patients were operated on under general anesthesia and 2 (4%) under sedative anesthesia. All patients were operated on with the Uniportal VATS technique. The postoperative treatment process was carried out with service follow-up without requiring intensive care follow-up. Twenty-eight (56%) patients were operated on from the right and 22 (44%) patients from the left hemithorax. When the pleural biopsy results were examined, malignancy was detected in 18 (36%) patients and tuberculosis in 8 (16%). Malignancies: 6 were adenocancer metastases (2 stomachs, 2 breast, and 2 colon cancers), 5 malignant mesotheliomas, 4 epidermoid cancer metastases (2 ovarian and 1 breast and 1 malignant melanoma), 2 lung adenocarcinoma infiltration, and 1 chondrosarcoma metastasis. No postoperative complications were observed in any of the patients.Conclusion: Uniportal VATS is an easy-to-apply, safe, effective, and rapid method for diagnosing pleural effusion.