Objectives: Aim of this study was to evaluate the accuracy and precision of the detection of individual miRNA as clinical biomarkers in the serum. Design and methods: miRNA-126 was quantified in serum using endogenous and exogenous controls fornormalization and the accuracy and precision of the method evaluated. The diagnostic value of serummiRNA-126 was evaluated in malignant esothelioma (MM) and non-small-cell lung cancer (NSCLC) patients using both relative and absolute qRT-PCR methods. Results: The use of endogenous invariant and exogenous synthetic controls as well sample dilution markedly improves the accuracy and precision of the assay. The inter- and intra-assay analyses revealed that relative qRT-PCR is a more reliable method. Circulating miR-126 detected in the serum by relative qRT-PCRs was found low-expressed in both malignancies, significantly differentiated MM patients from healthy controls and NSCLC from MM, but do not discriminate NSCLC patients from control subjects. Kaplan–Meier analysis revealed that low level of circulating miR-126 in MM patients was strongly associated with worse prognosis. Conclusions: We propose that this approach can be adopted for accurate analysis of other suitable circulating miRNA markers of different types of cancer.