Objectives: Malignant mesothelioma (MM) is the most serious asbestos-related disease. Its increasing incidence is alarming, suggesting the need for as early diagnosis as possible. This 4.5-year prospective longitudinal study aimed at assessing the benefit of measuring serum mesothelin as a marker for diagnosing malignant mesothelioma in individuals with previous occupational exposure to asbestos, as a part of their clinical follow-up care. Material and Methods: The study comprised 309 participants (235 males, 74 females) with a mean age of 58.9 years (standard deviation (SD) = 9.8) and a mean duration of exposure to asbestos dust of 13.4 years (SD = 9.3). From 2009 to June 2013, all subjects were followed at a department of occupational medicine in Olomouc. Apart from the standard parts of medical examination (history, physical examination, simple chest radiographs and spirometry), the patients’ serum mesothelin levels were determined by the Mesomark immunoenzymatic diagnostic assay. Statistical analysis of the validity of serum mesothelin level measurement was carried out with respect to the diagnosis of MM. Results: Among the participants, 16 (5.2%) individuals (14 males and 2 females) were diagnosed with malignant mesothelioma. Based on the detected mesothelin levels, their validity for prediction of malignant mesothelioma was calculated as follows: sensitivity – 0.75, specificity – 0.962, positive predictive value – 0.706, negative predictive value – 0.969, positive and negative likelihood ratios – 19.95 and 0.26, respectively, and diagnostic odds ratio – 76.8, at a 95% confidence interval. Conclusions: The high specificity was identified indicating the low false positivity as well. In the case of detecting elevated soluble mesothelin-related peptides (SMRP) levels in formerly asbestos-exposed individuals, the possibility of the presence of MM should be included into the clinical consideration. The high negative predictive value denotes a lower probability of the presence of MM in patients with normal SMRP levels but due to the limiting lower sensitivity this possibility cannot be entirely excluded.