Chemotaxis is an important aspect of immune cell behavior within the tumor microenvironment (TME). One prominent example of chemotaxis within the TME is the migration of regulatory T cells (Tregs) in response to the chemokine ligands CCL17 and CCL22. Tregs within the TME cause the suppression of anti-tumor immunity and inhibition of the effect of immunotherapeutic treatments. Therefore, the ability to screen for therapeutic antibodies that can inhibit or stimulate the chemotaxis of various immune cell types is crucial. Traditionally, chemotaxis is studied by determining the number of cells in the bottom reservoir of a Transwell microplate using flow cytometry; however, this method is time-consuming and thus not appropriate for high-throughput screening purposes. The Celigo Image Cytometer has been employed to perform high-throughput cell-based assays and was used to develop a new detection method for chemotaxis measurement. The image-based detection method was developed using chemokine ligands CCL17 and CCL22 to induce the migration of CCR4+ T cells and directly count them on the bottom of the Transwell plates. Finally, the method was applied to measure the inhibitory effects of commercially available anti-CCL17 and anti-CCL22 antibodies, which caused a dose-dependent decrease in the number of migrated T cells. The proposed image cytometry method allowed screening of multiple antibodies at various concentrations, simultaneously, which can improve the efficiency for discovering potential antibody candidates that can induce or inhibit recruitment of immune cells to the tumor microenvironment.