Aims: To improve RT-qPCR with an internal control and a synthetic standard curve to detect HEV in HIV co-infected patients. Methods and Results: A single-stranded RNA (ssRNA) and a double-stranded DNA (dsDNA) synthetic curve were designed, compared to the international reference panel for HEV genotypes, and tested to quantify and detect a reference panel for HEV genotypes. The detection limit of the RNA synthetic curve (50 copies per ml) was better than the DNA synthetic curve (100 copies per ml) and the WHO standard curve (250 copies per ml). Then, 280 serum samples from HIV-positive patients were tested for HEV RNA, which was detected in 3 6% of serum samples. The viral load ranged from 2 9 102 copies per ml to 4 78 9 108 copies per ml. HEV IgM/IgG antibodies were not detected in the RNA-positive patients. Sequencing analysis of HEV showed that the virus belongs to genotype 3 (HEV GT3). Conclusions: Real-time PCR was a useful tool to estimate co-infection with HEV/HIV, even in patients with low viral loads and undetectable anti-HEV IgG and IgM antibodies. Significance and Impact of the Study: Hepatitis E virus genotype 3 (HEV GT3) has been associated with silent chronic hepatitis and cirrhosis in HIVpositive subjects worldwide, but there is a lack of data on this co-infection in Brazil.