The study evaluates the suitability of miR-16 as a reference gene for normalization in serum-based microRNA analysis in melanoma patients. 143 primary cutaneous melanoma patients and 60 control subjects, including healthy volunteers, rheumatoid arthritis patients, non-melanoma cancer patients, and Atypical Mole Syndrome patients, were studied. MiR-16 expression was analyzed using qRT-PCR. While no significant difference was observed between melanoma patients and healthy volunteers, miR-16 expression varied significantly across stages of melanoma. The equivalence test failed to confirm equivalent expression of miR-16 in any melanoma versus control group pair. The results indicate that miR-16 is not a suitable reference gene for normalization in serum-based miRNA studies in melanoma patients.The study evaluates the suitability of miR-16 as a reference gene for normalization in serum-based microRNA analysis in melanoma patients. 143 primary cutaneous melanoma patients and 60 control subjects, including healthy volunteers, rheumatoid arthritis patients, non-melanoma cancer patients, and Atypical Mole Syndrome patients, were studied. MiR-16 expression was analyzed using qRT-PCR. While no significant difference was observed between melanoma patients and healthy volunteers, miR-16 expression varied significantly across stages of melanoma. The equivalence test failed to confirm equivalent expression of miR-16 in any melanoma versus control group pair. The results indicate that miR-16 is not a suitable reference gene for normalization in serum-based miRNA studies in melanoma patients.