The persistence of replication-competent HIV-1 in people living with HIV (PLWH) is a barrier to a cure for HIV. Early-phase studies of clinical interventions to deplete the intact persistent HIV-1 reservoir are ongoing. However, the ability to distinguish intact proviruses is limited by sequence variation and the predominance of defective proviruses. In this study, we developed HIVepsilon-seq (HIVe-seq), a novel assay to analyze 33 samples consisting of unfractionated peripheral blood mononuclear cells and/or resting CD4+ T cells from antiretroviral therapy (ART)-treated durably suppressed PLWH, including samples from 17 female participants. HIVe-seq combines simplified target enrichment and long-read sequencing methodology with advanced bioinformatic analysis to increase the depth, characterization, and detection of intact persistent HIV-1 provirus. HIVe-seq detected persistent sequence-intact HIV-1 proviruses in samples from both male and female participants and represents a robust, scalable assay to detect intact proviral sequences that could be applied to studies of HIV cure interventions. [ABSTRACT FROM AUTHOR]