Ben Berkhout studied molecular biology at Leiden University, and obtained his PhD in 1986 at the same university on a research project concerning the regulation of gene expression in RNA bacteriophages. He performed postdoctoral research at the Dana Farber Cancer Institute of the Harvard Medical School in the field of molecular immunology, and initiated his HIV-1 research at the National Institutes of Health (Bethesda).

Ben Berkhout initiated his own research line in 1991 upon return to the Netherlands, and has been at the Academic Medical Center (AMC) of the University of Amsterdam since then. He became Head of the Department of Human Retrovirology in 2002 and was appointed as Professor of Human Retrovirology in 2003. Furthermore, he is chairman of the AMC Research Institute for Infectious Diseases. He is editor of Journal of Biomedical Science and the open access journal Retrovirology, and associate editor for several journals (Journal of Virology, Nucleic Acids Research, Biotechnology, Journal of Biological Chemistry, Virology Journal, Journal of RNAi and Gene Silencing).