Background Late diagnosis of HIV fosters HIV transmission and may lead to hidden HIV epidemics. In Belgium, mathematical modeling indicates a high prevalence of undiagnosed HIV infections among men who have sex with men of non-Belgian origin and among sub-Saharan African migrants. Promotion of HIV testing facilitates early diagnosis, but diagnostic opportunities are missed in primary care. Objective The intervention study aims to enhance provider-initiated HIV testing by GPs. This protocol presents the conceptual development, implementation, and evaluation of an HIV-testing intervention for Flemish general practitioners (GPs). Methods A mixed methods evaluation design is used. Guided by a simplified intervention mapping approach, an evidence-based intervention was developed in collaboration, guided by an interdisciplinary advisory board. The intervention consisted of an evidence-based tool (ie, “HIV-testing advice for primary care”) to support GPs in provider-initiated HIV testing. A modified stepped-wedge design compare two different intervention levels: (1) online dissemination of the HIV-testing advice and (2) dissemination with additional group-level training. Both conditions were compared against a control condition with no intervention. The effect of the intervention was measured using Poisson regression for national surveillance data. The primary outcome was the number of HIV diagnoses made by GPs. Secondary outcomes were HIV diagnoses among groups at risk for undiagnosed HIV, distribution of new diagnoses by CD4 cell count, number of HIV tests prescribed by GPs, and rate of new diagnoses by tests. To evaluate the intervention’s implementation, the GPs’ fidelity to the intervention and the intervention’s feasibility and acceptability by GPs were assessed through (web-based) surveys and in-depth telephone interviews. Results The study was funded in 2016 and ethically approved in January 2017. The implementation of the intervention started in January 2017 and ended in December 2018. Data was completed in October 2019 and was the starting point for the ongoing data analysis. The results are expected to be published in the second half of 2020. Conclusions Results of the intervention study will provide useful information on the intervention’s effectiveness among Flemish GPs and can inform further development of official testing guidelines. Limitations of this real-life intervention approach are potential spill-over effects, delay in access to surveillance data, and little detailed information on HIV-testing practices among GPs. Trial Registration ClinicalTrials.gov NCT04056156; https://clinicaltrials.gov/ct2/show/NCT04056156 International Registered Report Identifier (IRRID) DERR1-10.2196/16486