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Asbestosis and mesothelioma are diseases caused by inhalation of asbestos dust during working in the mines or factories. The tentative diagnosis of most cases is usually based on circumstantial evidence, which might not be accurate unless asbestos materials are pathologically confirmed at the lesions available. In other cases of substantiation of asbestos exposure, tests for biomarkers such as serum tropomyosin 4 (TPM4) and peroxiredoxin II (PRXII or PRDX2) have been employed. In this regard, we conducted experiments to develop two in-house sandwich ELISA kits for determining human serum TPM4 and PRXII. We used bioinformatics evaluation to find antibody pairs from 5 commercial antibodies. We optimized for the appropriate antibody concentrations, standard diluents, blocking solutions, and created an appropriate standard curve for each biomarker. There were two capture-detection antibody pairs for TPM4 and PRXII which were selected for developing the test kits, namely: TPM4 (E-17) and TPM4 (OAAB09038), and PRXII (N-13) and PRDX2 (OABB00785). The invented test kits for detection of serum TPM4 and PRXII were shown to lower limits for detection and the detection ranges were comparable to those reported in commercial kits. Further, the efficacy of the two invented kits’ was comparable to commercial kits and the cost for running the kits is affordable.