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Abstract Background The survival of HIV-infected infants depends on early identification and initiation on effective treatment. HIV-exposed infants are tested at 6 weeks of age; however, testing for HIV sooner (e.g., shortly after birth) can identify in utero infection, which is associated with rapid progression. Infant early diagnostic virologic tests often have long turnaround times, reducing the utility of early testing. Point-of-care (POC) testing allows neonates born in health facilities to get results prior to discharge. This study aimed to understand the views of mothers and health workers regarding the use and acceptability of POC birth testing. Methods Beginning in 2018, Zimbabwe offered standard HIV testing at birth to high-risk HIV-exposed infants; as part of a pilot program, at 10 selected hospitals, POC birth testing (BT) was offered to every HIV-exposed infant. In order to understand experiences at the selected sites, 48 interviews were held: 23 with mothers and 25 with health workers, including 6 nurses-in-charge. Participants were purposively sampled across the participating sites. Interviews were held in English, Shona, or Ndebele, and transcribed in English. Line-by-line coding was carried out, and the constant comparison method of analysis was used to identify key themes for each respondent type. Results Findings were organized under four themes: challenges with BT, acceptability of BT, benefits of BT, and recommendations for BT programs. Overall, BT was well accepted by mothers and health workers because it encouraged mothers to better care for their uninfected newborns or initiate treatment more rapidly for infected infants. While the benefits were well understood, mothers felt there were some challenges, namely that they should be informed in advance about testing procedures and tested in a more private setting. Mothers and HCWs also recommended improving awareness of BT, both among health care workers and in the community in general, as well as ensuring that facilities are well-stocked with supplies and can deliver results in a timely way before scaling up programs. Conclusions Mothers and health workers strongly support implementation and expansion of birth testing programs due to the benefits for newborns. The challenges noted should be taken as planning guidance, rather than reasons to delay or discontinue birth testing programs.