Objective The objective was to assess the efficacy of nucleic acid amplification testing (NAAT) in conjunction with serology for detecting hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV) in donor blood products. Methodology This retrospective cross-sectional study aimed to evaluate the use of NAAT and viral serology in screening donor blood products for HBV, HCV, and HIV. The study was conducted at the Dr Ishrat-ul-Ebad Khan Khan Institute of Blood Diseases at Dow University of Health Sciences in Karachi, Pakistan, from April 2018 to May 2022. All blood products, after preparation, were screened for HBV, HCV, and HIV types I and II using electrochemiluminescence immunoassay on the Roche cobas e 601 analyzer (Roche, Basel, Switzerland). For further confirmation, blood products with negative viral serology underwent NAAT using the cobas TaqScreen MPX test version 2.0 (Roche) on the cobas s 201 system (Roche) to detect the window period of replicating viruses. Simple calculations were performed to determine the NAAT yield and yield rate for each virus, as well as the total number of viruses in seronegative donors. Results A total of 46,455 donors visited the Dr Ishrat-ul-Ebad Khan Khan Institute of Blood Diseases for blood donation. Of these, 6.97% (n = 3,240) tested positive for HIV, HBV, or HCV during serology screening, making them ineligible for donation. The remaining 93.02% (n = 43,215) of seronegative donors underwent NAAT to detect infections during the window period. NAAT revealed a reactivity of 0.044% (n = 19) for HBV, 0.009% (n = 4) for HCV, and 0.00% (n = 0) for HIV. The total NAAT yield rate for HBV was 1 in 2,252, and for HCV, it was 1 in 11,111. The overall NAAT yield rate for all viruses was 1 in 1,886 (0.053%) donor blood products. Conclusions These findings highlight the effectiveness of NAAT in identifying blood-borne infections during the window period. Consequently, routine NAAT screening for all seronegative blood donors is a crucial step that can reduce the burden of transfusion-transmitted infections and enhance the safety of blood products, especially in underdeveloped countries like Pakistan.