The aim of the present study was to assess the feasibility of a 3D tumor cell culture model, that is, multicellular tumor spheroids (MCTSs) as an adequate model for micrometastases and therefore as a pharmacological model for efficacy testing of trifunctional therapeutic antibodies. Unlike conventional monolayer cultures, spheroids allow researchers to study parameters, such as 3D cell shape, 3D cell arrangement and microenvironment, and penetration efficiency of defense cells that may largely influence the efficacy of antibody treatment in vivo. The authors established a long-term coculture of human MCTSs with peripheral blood mononuclear cells (PBMCs) to test the anticancer effect of the trifunctional, bispecific antibody catumaxomab (anti-EpCAM x anti-CD3) or similar therapeutic molecules. The test system is accessible to various analytical methods and thus allows for characterizing multiple parameters, which can help elucidate the mode of action of immunotherapeutic anticancer treatment. For example, the novel approach enables precise, reproducible volume growth analysis of MCTSs under immunotherapeutic treatments. For evaluation of changes within individual spheroids, cryosections can be stained (e.g., for proliferating or apoptotic cells as well as infiltrating PBMCs). Molecular PCR-based assays or flow cytometric analyses allow for discrimination between different cell types, particularly leukocyte subtypes. Furthermore, MCTSs can be disaggregated to form standard monolayers for cell viability or plating efficiency experiments. For these reasons, the MCTS model is a powerful tool to analyze drug efficacy with various endpoints under highly reproducible, standardized conditions.