This study evaluated the feasibility, safety, and acceptability of self-collected vaginal samples for human papillomavirus (HPV) testing among pregnant women in an urban teaching hospital in Malaysia. This cross-sectional study recruited pregnant women aged 30 years and above who attended antenatal care at the University of Malaya Medical Centre. Participants self-collected vaginal samples using FLOQSwabs and completed pre- and post-sampling questionnaires assessing acceptability. Samples were analysed using the Roche cobas 4800 system. HPV-positive participants were referred for postpartum colposcopy. A total of 2,256 eligible pregnant women were invited to participate in the study and 1,603 of them consented to participate, representing 71.1% of uptake. Of the 1,603 participants, 99.6% (1,596) agreed to self-collect, with 98.6% successfully completing the procedure. More than 80% of participants responded positively to acceptability indicators after self-collection procedure, including overall feeling, ease of performance, convenience, and confidence in collecting an accurate sample. The HPV prevalence recorded in the study population was 6.3%, with most infections involving non-HPV16/18 types. No major complications associated with the sampling procedure were reported, and 99.0% of participants expressed willingness to use self-collection for future cervical screening. Self-collection for HPV-based cervical screening during pregnancy is highly acceptable, safe, and feasible, with performance comparable to the general population. Integrating self-collection into antenatal care can enhance cervical cancer screening rates among under-screened populations, contributing to global elimination goals.