Pleural effusion (PE) is excess fluid that appears in the pleural cavity of many lung cancer patients, as well as in those suffering from other ailments like extra-pulmonary tuberculosis (TB) and pneumonia, or stems from a variety of more benign diseases. Diagnosing the cause of the PE is often a clinical challenge and we have applied targeted proteomic methods with the aim of aiding the determination of PE etiology. We developed a mass spectrometry (MS)-based multiple reaction monitoring (MRM)-protein-panel assay to precisely quantitate 53 established cancer-markers, TB-markers, and infection/inflammation-markers currently assessed individually in the clinic, as well as potential biomarkers suggested in the research literature for PE classification. Since MS-based targeted proteomic assays are on the cusp of entering clinical use, we assessed the merits of such an approach and this marker panel in classifying PEs based on a single-center 209 patient cohort with established etiology. We observed groups of infection/inflammation markers (ADA2, WARS, CXCL10, S100A9, VIM, APCS, LGALS1, CRP, MMP9, and LDHA) that specifically discriminate TB-PEs and other-infectious-PEs, and a number of cancer markers (CDH1, MUC1/CA-15-3, THBS4, MSLN, HPX, SVEP1, SPINT1, CK-18, and CK-8) that discriminate cancerous-PEs. Some previously suggested potential biomarkers did not show any significance. Using a Decision Tree/Multiclass classification method, we show a very good discrimination ability for classifying PEs into one of four PE types: AUC of 0.863 for cancerous-PEs, AUC of 0.859 for tuberculous-PEs, AUC of 0.863 for other-infectious-PEs, and AUC of 0.842 for benign-PEs. Such multiplexed MS assays and the indicated markers show future potential in aiding clinical diagnosis after standard imaging techniques and thoracentesis to help in the hard decision on therapy guidance.