An all-in-one (AIO) dry-reagent time-resolved fluorometric immunoassay that requires minimal liquid handling was developed for the detection of anti-HIV-1 and -2 antibodies. To prepare the AIO wells, in vivo biotinylated capture antigens (r-Bio-HIV-1 env and r-Bio-HIV-2env) were immobilized on streptavidin-coated microtitration wells and Eu(III) chelate labelled non-biotinylated tracer antigens [r-HIV-1env-Eu(III) and r-HIV-2env-Eu(III)] were dried in stable form in the same wells. The HIV MO assay was evaluated with serum/plasma samples (n = 148) from in-house and commercial panels at two different incubation times of 15 min and 1 h. The overall sensitivity of the AIO assay was 98.6% and specificity was 100% for both the incubation times. The AIO assay can accept whole blood matrix. This assay is envisioned to fill the gap between the rapid point-of-care assays and traditional enzyme immunoassays (EIA) in terms of complexity and turnaround time, without compromising the performance. (C) 2015 Elsevier B.V. All rights reserved.