A study was conducted to assess the efficacy of various DNA extraction methods for detecting viral DNA in newborn screening cards (NSC). The aim was to identify potential viral infections during pregnancy and birth. Seronegative and seropositive blood samples were spotted onto blank NSC and subjected to DNA extraction using commercial and noncommercial methods. The extracted DNA was quantified using the PicoGreen dsDNA quantification kit, and amplification protocols were evaluated using serial dilutions of purified viral DNA controls. The sensitivity of the extraction methods was compared, with the QIamp DNA micro kit with forensic filter showing marginally better sensitivity compared to noncommercial methods (P=0.014; P=0.016). This method yielded a mean DNA yield of 3 ng/l, and a 50-liter reaction could detect 10 copies of viral genome. DNA extracts diluted 1:10 and neat were found to be reliable for Epstein-Barr virus (EBV) nested PCR detection. The study concluded that the QIamp Micro DNA extraction method, in combination with a forensic spin-filter step, was effective for retrospective DNA viral assays from NSC.