BACKGROUND: Persistent latent reservoirs of intact HIV-1 proviruses, capable of rebounding despite suppressive antiretroviral therapy (ART), hinder efforts towards an HIV-1 cure. Hence, assays specifically quantifying intact proviruses are crucial to assess the impact of curative interventions. Two recent assays have been utilized in clinical trials: intact proviral DNA assay (IPDA) and quadruplex quantitative PCR (Q4PCR). While IPDA is more sensitive due to amplifying short fragments, it may overestimate intact fractions by relying only on quantification of 2 proviral regions. Q4PCR samples 4 proviral regions, yet is sequencing-based, favoring amplification of shorter, hence non-intact, proviral sequences. METHODS: Leveraging digital PCR (dPCR) advancements, we developed the "Rainbow" 5-plex proviral HIV-1 DNA assay. This first-in-its-kind assay was evaluated using standard materials and samples from 83 people living with HIV-1, enabling simultaneous quantification of both total and intact HIV-1 DNA levels. HIV proviral unique molecular identifier (UMI)-mediated long-read sequencing (HIV-PULSE) was used to validate the specificity of the Rainbow HIV-1 DNA assay. RESULTS: The Rainbow assay proved equally sensitive but more specific than IPDA and is not subjected to bias against full-length proviruses, enabling high-throughput quantification of total and intact reservoir size. The near full-length sequences allowed validation of the Rainbow specificity and the design of personalized Rainbow primer/probe sets, which enabled the detection of intact HIV-1 DNA. CONCLUSIONS: This innovation offers potential for targeted evaluation and monitoring of potential rebound-competent reservoirs, contributing to HIV-1 management and cure strategies. ClinicalTrials.gov Registration Numbers: NCT04553081, NCT04305665.