The validation of virus-free blood donors is often neglected possibly related to the high cost process analyzing. While, validity of the medical status of hepatitis C virus (HCV) is crucial for blood donors quality. ELISA is common method for HCV detection, however inaccurate result possibly occurred during the window period. Hence, the validation of this method using molecular test is still required. The samples were collected from 100 blood donors consist of 85 males (85%) and 15 females (15%) from Tuban region, East Java Indonesia. The ELISA kit test resulted 100% of negative HCV. Surprisingly, after validated using PCR there were found 6 blood samples (6%) positively infected HCV. This study encourages who one concerned to propose that PCR-based validation for screening blood donors is necessarily required.