BACKGROUND:Improved assays are critical to better characterize the HIV reservoir and to reliably evaluate candidate intervention strategies. Here we describe different methods to quantify the HIV reservoir. METHODS:We developed an optimized quantitative viral outgrowth assay (QVOA) to quantify the frequency of cells harboring replication-competent HIV, which is simpler and more sensitive than classical QVOAs. We also developed new inducible RNA assays that concomitantly measure the frequency of cell-associated [ca-] (gag and tat-rev) and cell-free [cf-] HIV RNA after three days of anti-CD3/CD28 stimulation. FINDINGS:The median frequency of the infected cells measured after induction was 94 IQR[60-132], 16 IQR [9-29] and 2.9 IQR[1.9-6.8] cells/106 CD4+ T-cells for ca-RNA gag and tat-rev, and cf-RNA, respectively. There are a large proportion of transcription-competent proviruses (ca-RNA) that seemed unable to form complete virions (cf-RNA), suggesting post-transcriptional blocks or defective proviruses. Importantly, the median frequency of infected CD4+ T-cells as estimated by 3-day inducible cf-RNA assay was not statistically different from the frequency measured by the QVOA (median of 3.3 [1.9-6.2] IUPM). The latently infected cells detected by the inducible cf-RNA assay correlated highly with the QVOA ( r= 0.67, p < .001), and both assays were equivalent in 60% of the samples tested, suggesting that most cells induced to produce virions are generating replication-competent virus. INTERPRETATION:These inducible RNA assays provide more sensitivity and a greater dynamic range for the monitoring of reduction of the reservoir by eradication strategies. Such assays may serve as robust and useful tools for clinical investigations of the HIV reservoir.