The efficacy and mechanism of action of therapeutic antibodies that target cancer cells have typically been evaluated using in vitro assays and long-term in vivo tumor models. To allow for a more efficient assessment of the function of candidate therapeutic antibodies, we have developed a flow cytometric-based method that rapidly and directly quantifies antibody-mediated killing in a short term in vivo assay. Target cells that express human CD52, including huCD52(+) splenocytes from huCD52 transgenic mice and Ramos cells, a CD52(+) human B cell lymphoma line, and CD52(-) reference cells were differentially labeled by using two fluorescent dyes to distinguish target and reference cell populations. Labeled cells were injected into mice with or without Campath-1H (Alemtuzumab) and then recovered for flow cytometric analysis 5 h later. We found that huCD52(+) transgenic splenocytes and Ramos cells were selectively depleted in Campath-treated animals but not in animals treated with a negative control antibody. Furthermore, it is likely that the cells were depleted in vivo by a complement-dependent mechanism since target cell depletion was significantly reversed after complement inactivation using cobra venom factor. This report demonstrates the feasibility and utility of a powerful method for the rapid evaluation in vivo of therapeutic antibody candidates for cancer.