Background:Although the sensitivity of third generation anti-HCV CIA tests is high, false positivity rates, especially in populations with low HCV infection endemicity, are still high. Objectives:We aimed to determine the S/Co cut-off value of anti-HCV in the diagnosis of real positive patients based on the CIA test kit absorbance routinely used in our laboratory and to reveal the potential cost effectiveness of confirmatory tests for false positive samples. Methods:All anti-HCV CIA test results which were performed in the microbiology laboratory of our hospital between 2016-2019 were retrospectively screened and S/Co values of the patients were recorded. Among these, the results that were confirmed with HCV-RNA real-time PCR test were included. Patients who were previously diagnosed and treated were excluded. Results:A total of 257 patients, who were tested for HCV-RNA after reactive anti-HCV test results, were included in the study. Of the cases, 84(32.68%) had positive HCV-RNA. According to the ROC analysis, the optimal S/Co value was 8.58 with the sensitivity and specificity values 95.24% and 85.55%, respectively. According to this 8.58S/Co value, anti-HCV test was reactive in 105 cases and 80(76.2%) of these cases had active HCV infection. In order to prevent the false-negativity, the additional cost of using 1.0S/Co value to our institution was 4114.64USD, meaning that we spent 1028.66USD to diagnose per true-case of active HCV infection when using 1.0S/Co value. In our institution, approximately 6.25 working hours are spent to finalize the HCV-RNA PCR test. The hours spent for S/Co of 1.0 and 8.58 were 1606.25 and 658.25, respectively. Conclusions:False positive anti-HCV results are an economic burden on health economics of countries. At least, different S/Co values might be used in accordance with the purpose of the screening (like blood donors or pre-operative screening) and prevalence of HCV infection in different laboratories and different populations.