BACKGROUND: HIV disproportionately affects women of childbearing age in South Africa. Despite progress in antiretroviral therapy access, challenges persist in HIV vertical transmission prevention (VTP). METHODS: We conducted a single-centre, retrospective observational audit of women delivering between May and August 2023 at the Charlotte Maxeke Johannesburg Academic Hospital in Johannesburg, South Africa to assess adherence to national VTP guidelines. RESULTS: Of 563 women, 22% (125/563) were living with HIV at their first antenatal visit, increasing to 24.3% (137/563) by delivery. HIV retesting rates declined from 46% (181/394) at the second visit to 20% (30/151) by the eighth. HIV viral load testing was performed in 71.5% of women (98/137) at delivery, with 72.4% (71/98) achieving viral suppression. Birth PCR testing was not performed for 21.1% (29/137) of HIV-exposed infants. CONCLUSION: Significant gaps in HIV retesting, viral load monitoring, and birth PCR testing highlight ongoing challenges in VTP guideline adherence.