Background: We have verified a mass spectrometry (MS)–based targeted proteomics signature for the detection of malignant pleural mesothelioma (MPM) from the blood. Methods: A seven-peptide biomarker MPM signature by targeted proteomics in serum was identified in a previous independent study. Here, we have verified the predictive accuracy of a reduced version of that signature, now composed of six-peptide biomarkers. We have applied liquid chromatography–selected reaction monitoring (LC-SRM), also known as multiple-reaction monitoring (MRM), for the investigation of 402 serum samples from 213 patients with MPM and 189 cancer-free asbestos-exposed donors from the United States, Australia, and Europe. Results: Each of the biomarkers composing the signature was independently informative, with no apparent functional or physical relation to each other. The multiplexing possibility offered by MS proteomics allowed their integration into a single signature with a higher discriminating capacity than that of the single biomarkers alone. The strategy allowed in this way to increase their potential utility for clinical decisions. The signature discriminated patients with MPM and asbestos-exposed donors with AUC of 0.738. For early-stage MPM, AUC was 0.765. This signature was also prognostic, and Kaplan–Meier analysis showed a significant difference between high- and low-risk groups with an HR of 1.659 (95% CI, 1.075–2.562; P ¼ 0.021). Conclusions: Targeted proteomics allowed the development of a multianalyte signature with diagnostic and prognostic potential for MPM from the blood. Impact: The proteomic signature represents an additional diagnostic approach for informing clinical decisions for patients at risk for MPM.