Cell to cell transmission of HIV has been proposed as a mechanism contributing to virus escape to the action of antiretrovirals and a mode of HIV persistence during antiretroviral therapy. Here, cocultures of infected HIV-1 cells with primary CD4+ T cells or lymphoid cell lines were used to evaluate virus transmission and the effect of known antiretrovirals. Transfer of HIV antigen from infected to uninfected cells was resistant to the reverse transcriptase inhibitors (AZT and tenofovir), but was blocked by the attachment inhibitor IgGb12. However, quantitative measurement of viral DNA production demonstrated that all anti-HIV agents blocked virus replication with similar potency compared to cell-free virus infections. Similarly, cell-free and cell-associated infections were equally sensitive to inhibition of viral replication when measuring HIV-1 LTR-driven GFP expression in target cells. However, detection of GFP by flow cytometry may incorrectly estimate the efficacy of antiretrovirals in cell-associated virus transmission, due to replication independent Tat-mediated LTR transactivation consequence of cell-to-cell events that did not occur in short-term (48h) cell-free virus infections. In conclusion, common markers of virus replication may not accurately correlate and measure infectivity or drug efficacy in cell to cell virus transmission. When accurately quantified, active drugs blocked proviral DNA and virus replication in cell to cell transmission, recapitulating the efficacy of antiretrovirals in cell-free virus infections and in vivo.