A method of determining the latent HIV reservoir level in a subject includes obtaining a blood sample from an HIV+ subject, isolating CD4+ T cells from the biological sample, administering one or more HIV transcription inducing agents to the isolated CD4+ T cells, isolating RNA from the CD4+ T-cells that includes HIV env mRNA, producing a plurality of first amplicons from the from the isolated RNA using a first primer set that corresponds to an HIV genomic region encoding the HIV env protein, producing a plurality of second amplicons from the plurality of first amplicons using a second primer set, the second primer set including one or more adapter sequences and/or uniquely identifiable barcode sequences, and determining the nucleic acid sequences of the second amplicons, wherein the determined nucleic acid sequences in the sample are indicative of the amount of inducible cell-associated HIV env RNA in the sample and indicative of the latent HIV reservoir in the subject.