The diagnosis of hepatitis B virus infection is evaluated serologically, virologically, biochemically, and with histologic liver indicators. The aim of this study was to investigate the place and significance of the HBsAg neutralization test in the diagnostic algorithm for hepatitis Binfection. From the venous blood samples sent to Ankara Numune Education and Research Hospital Medical Microbiology Laboratory between September 2014 and May 2016 for HBsAg test, serum samples regarded each patient as reactive (≥ 0.9 S\CO) , 9 ≤ S ≤ CO ≤ 30) were studied twice. A total of 105 samples which were reactive in both analyses were included in the study. After the evaluation of these samples by neutralization confirmation test, which is routinely performed in our laboratory, the samples were stored under optimal conditions and studied for HBV DNA with the real-time PCR and for HBeAg, anti-HBeAg, anti-HBc IgM, and anti-HBc total antibody assays by ELISA. The 105 samples, in which HBsAg was detected, were analyzed with the neutralization test. The presence of HBsAg was confirmed by neutralization test in 67 of 105 samples (63.8%), and of these patients, two patients (2.3%) had negative HBV DNA and anti-HBc total antibody test (false positive neutralization test). Of the 105 samples included in the study, the anti-HBc total antibody test was positive in 78 patients (74.3%). However, out of these 78 patients who were positive for the anti-HBc total antibody test, there were 13 patients (16.7%) with negative neutralization and HBV DNA test results (false positive anti-HBc total antibody test). The HBV DNA positivity was detected significantly lower in samples with HBsAg level ≤ 5 S/CO compared to the samples with HBsAg level > 5 S/CO (p= 0.020). Also, if the unit price of the neutralization test used in our study was considered, the cost was 17,00 TL while the unit price of HBV DNA test was 55,00 TL. Utilization of the neutralization test instead of HBV DNA test provides a saving of 38,00 TL per patient. The use of the neutralization test as a validation test when the HBsAg titre was less than or equal to 5S/CO will significantly reduce the cost of the test without the need for HBV DNA test. We suggest that if both the neutralization test and the anti-HBc total antibody test is negative in a sample with an HBsAg titration higher than 5 S/CO, the decision can be made without the need for HBV DNA test. However; if the neutralization test is negative, but the anti-HBc total antibody test is positive, then HBV DNA needs to be determined to eliminate the inconsistency between neutralization and anti-HBc total antibody assays. At the same time, we found that the use of anti-HBc total antibody test along with HBsAg as a screening test did not provide any advantage since the anti-HBc total antibody test was detected to have a high false positivity (16.7%) rate.