The aim of this study was to determine the clinical benefit of a new combined antigen-antibody immunoenzymatic assay (Monolisa HCV Ag-Ab Ultra, Biorad) in the setting of acute HCV infection in HIV infected patients. The performance of this assay was first evaluated in 160 HIV positive samples from uninfected and chronically HCV infected patients. To assess the performance of the Ag-Ab assay in the context of acute hepatitis C, 94 stored frozen serums from 20 recently diagnosed cases were retrospectively tested for HCV-RNA and presence of anti-HCV antibodies, in parallel with the new assay. In HIV infected patients, the sensitivity and specificity of the Ultra assay was 100% with a strong discrimination between positive and negative samples. In HCV acutely infected patients, the Ag-Ab assay significantly reduced the seronegative period, allowing an earlier diagnosis compared to a 3rd generation ELISA in 65% of the cases. The combined assay became positive on the same bleed as the first HCV-RNA detection for 13 patients out of 20. Nevertheless, in one case, characterized by an absence of seroconversion over one year but a continuous viral replication above 1 million IU/ml, the combined assay did not improve HCV infection diagnosis. Use of this new assay as a first line screening would significantly reduce the long seronegative window period seen in HCV infection allowing earlier HCV diagnosis and rapid clinical management. However, in case of clinical acute hepatitis C, sensitive HCV-RNA detection should remain the gold standard.