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To facilitate the translation of immunotherapies from bench to bedside, predictive preclinical models are essential. We developed the in vivo immuno-oncology Hollow Fiber Assay (HFA) to bridge the gap between simpler cell-based in vitro assays and more complex mouse models for immuno-oncology drug evaluation. The assay involves co-culturing human cancer cell lines or primary patient-derived cancer cells with human immune cells inside semipermeable hollow fibers. Implanted intraperitoneally in mice, the fibers captured treatmentinduced immune cell-mediated cancer cell killing following treatments with aCD3 and/or IL-2, demonstrating the feasibility of the assay. Traditional models require lengthy observation periods to monitor tumor growth and treatment response. The immuno-oncology HFA enables a rapid initial in vivo evaluation of immunological agents on cancer and immune cells of human origin, addressing two of the 3Rs - reduction and refinement - in animal research.