[group B (GBS)] poses a major threat as the primary cause of early-onset neonatal invasive disease, particularly when mothers are colonized rectovaginally. Although culture remains the gold standard for antepartum GBS screening, quantitative polymerase chain reaction (qPCR) offers advantages in terms of sensitivity and turnaround time. The aim of this study was to validate the clinical utility of an automated qPCR laboratory-developed test (LDT) for antepartum GBS screening using the Panther Fusion Open Access system (Hologic, California, USA). The LDT targeted a conserved region of the GBS gene, demonstrating no cross-reactivity and high coverage (99.82%-99.99%). The limit of detection (LoD) was 118 CFU/mL. Comparison with commercial qPCR assays (Panther Fusion GBS and VIASURE B Real-Time) revealed an overall agreement of 99.7%, with a robust Cohen's kappa coefficient of 0.992. Testing of 285 rectovaginal swabs from pregnant women and 15 external quality assessment samples demonstrated exceptional diagnostic performance of the LDT, achieving a diagnostic sensitivity and specificity of 100%, underscoring its accuracy. Prevalence and predictive values were also determined to reinforce test reliability. Our research highlights the limitations of culture-based screening and supports the suitability of our qPCR-based LDT for GBS detection in a clinical setting.IMPORTANCERectovaginal colonization by GBS is a major risk factor for early-onset invasive neonatal disease. The most effective approach to reducing the incidence of early-onset disease (EOD) has been described as universal screening, involving assessment of GBS colonization status in late pregnancy and intrapartum antibiotic prophylaxis. Despite its turnaround time and sensitivity limitations, culture remains the gold standard method for GBS screening. However, nucleic acid amplification-based tests, such as qPCR, have been utilized due to their speed and high sensitivity and specificity. This study validated the clinical usefulness of an automated qPCR-LDT for antepartum GBS screening through the Panther Fusion Open Access system (Hologic). Our study addresses the critical need for more robust, sensitive, and rapid strategies for GBS screening in pregnant women that could favorably impact the incidence of EOD.