The World Health Organization has realized Option B + program to ensure lifelong antiretroviral therapy for all women with human immunodeficiency virus to eradicate the vertical transmission of the virus. The Option B + program is expected to achieve the United Nations target of 95% retention in care by 2025. Despite the successful implementation of Option B + in southern Ethiopia for the past nine years, the issue of loss to follow-up continues to be a major challenge. Thus, this study aims to examine the incidence and determinants of loss to follow-up in women under the Option B + program in southern Ethiopia from 2014 to 2022. A retrospective cohort study was conducted, and data were collected using a structured checklist from 468 charts of women on Option B+, of which 441 were included in the final analysis. The Kaplan-Meier survival curve with the Log-rank test was used to estimate the survival time. Bivariable and multivariable Cox proportional hazard regression models were fitted to identify determinants of loss to follow-up. Overall, 17.5% of women were lost to follow-up during the entire observation period, resulting in 5.9 new cases per 1000 women in one month (95% CI: 4.8–7.5). The cumulative probability of loss to follow-ups increased over time, from 2.7% at 6 months to 4.2% at 12 months, and peaked at 43.4% at 60 months. The multivariable Cox regression results revealed that women at a higher risk of loss to follow-up were those who received antiretroviral therapy at health centers compared to those enrolled at hospitals, initiated antiretroviral therapy on the day of diagnosis compared to those who started later, had poor antiretroviral therapy adherence, and did not disclose their serostatus to anyone. The rate of loss to follow-up in southern Ethiopia is not on track to meet the United Nations target. Therefore, it is crucial to develop practical strategies for enhancing antiretroviral therapy adherence, considering individual factors for optimal treatment timing, evaluating the quality of health centers’ antiretroviral therapy services, and addressing women’s concerns about disclosure to minimize loss to follow-up.