Background: Programmed cell death protein 1 (PD-1)/programmed cell death protein ligand 1 (PD-L1) immune checkpoint inhibitors have been approved for monotherapy of metastatic nonsmall cell lung cancer (mNSCLC) depending on tumour cells' PD-L1 expression. Pleural effusion is common in mNSCLC. The significance of immunocytochemistry PD-L1 analysis from pleural effusion samples is unclear. Aim: The aim of the study was to analyse the sensitivity regarding immunocytochemistry PD-L1 analysis of pleural effusion in NSCLC as compared to immunohistochemistry of pleural biopsies. Patients and Methods: Fifty consecutive subjects (17 female, median age 72.5 years, seven never-smokers) were enrolled in this prospective controlled two-centre study. Inclusion criteria were pleural effusion, suspected or known lung cancer, indication for pleural puncture and thoracoscopy, and written informed consent. Immunocytochemistry and immunohistochemistry PD-L1 analyses were performed with the Dako-PDL1-IHC-22C3pharmDx assay. Analysis for sensitivity, specificity, and positive and negative predictive value was performed for PD-L1 detection from pleural effusion. Results: 50 subjects underwent pleural puncture and thoracoscopy. Pathological diagnoses were lung cancer (48), lymphoma (1) and mesothelioma (1). Sensitivity, specificity, positive predictive value and negative predictive value of PD-L1-testing with expression ≥50% defined as positive were 100% (95% CI 46–100%), 63% (36–84%), 45% (18–75%) and 100% (66–100%), and with expression ≥1% defined as positive 86% (56–97%), 43% (12–80%), 75% (47–92%) and 60% (17–93%). Conclusion: PD-L1 analysis in tumour-positive pleural effusion samples shows a very high sensitivity and negative predictive value, especially regarding PD-L1 expression levels ≥50% (European Medicines Agency approval). Negative results are reliable and help in the decision against a first-line checkpoint inhibitor monotherapy. However, a 1% cut-off level (United States Food and Drug Administration approval) leads to a markedly lower negative predictive value, making other invasive procedures necessary (NCT02855281).