Technical Abstract: Covalent binding of biotin to histones participates in heterochromatin formation, cell cycle progression, and the cellular response to DNA breaks. Biotinylation of histones appears to be a reversible process but the identities of enzymes that remove the biotin mark are largely unknown. Our long-term goal is to identify histone debiotinylases in human cells. Here we developed an avidin-based plate assay to quantify histone debiotinylase activities in human cell nuclei. This assay is an essential first step in purifying histone debiotinylases from human cell nuclei. Using this assay we demonstrated that debiotinylation of human histones is a temperature and pH-dependent process, consistent with enzyme catalysis. Studies with purified proteases and competitiors suggested that removal of the biotin mark in histones is mediated by debiotinylases rather than proteases. Activities of histone debiotinylases varied among human tissues: colon = lung > placenta = liver > lymphoid cells. The assay proved useful to monitor activities of semi-purified histone debiotinylases from human body fluids.