In recent years, the prediction of oral drug absorption in humans has been a challenge for researchers and many techniques for permeability studies have been developed for several purposes, including biowaiver processes. The Single-Pass Intestinal Perfusion (SPIP) method performed in rats can provide permeability results closest to in vivo condition. The purpose of the present study was to evaluate the intestinal permeability of the antiretroviral drugs lamivudine, stavudine and zidovudine using the SPIP method in rats and to predict their permeability (Peff,humans) and fraction absorbed (Fa) in humans. Metoprolol and fluorescein were used as marker compounds of high and low permeability, respectively. The effective permeability (Peff) results showed that stavudine and zidovudine have high permeability characteristics while lamivudine presented the lowest result. From Peff values obtained in rats, the Peff,humans and Fa were calculated. The use of SPIP in rats and calculations for absorption prediction in humans may indicate the transport mechanisms and/or pre-systemic metabolism involved on permeation processes of drugs, since this model is the closest to in vivo conditions.