Introduction: Approximately a quarter of people living with HIV (PLHIV) had their plasma viral load (PVL) measured in 2020 in Burkina Faso. The purpose of this study was to assess the knowledge, attitudes, and practices (KAP) of health workers regarding HIV PVL measurement. Methods: A cross-sectional study was conducted among health workers involved in the care of PLHIV in the 13 regions of Burkina Faso in 2021. Scores were constructed to assess their KAP on PVL measurement. Factors associated with knowledge and practices were identified by logistic regression. Results: A total of 255 health workers were surveyed. The majority had good knowledge (73%) and favorable attitudes (93%). However, 40% had inadequate practices. Taking into account the availability of a laboratory to carry out PVL tests within the health center, having a coordinating role increased the likelihood of having good knowledge, while not having a medical qualification reduced this likelihood. Good practices were more common among health workers working at the second level of the health pyramid. Conclusions: Interventions to increase the demand for a measurement of PVL are essential to improve the care of PLHIV. For example, future investigations could explore the role of mediators in increasing the demand for PVL among PLHIV and their caregivers.