Macrophage migration inhibitory factor (MIF) is a pleiotropic inflammatory cytokine and atypical chemokine that regulates both innate and adaptive immunity. Its broad expression and stress-induced release make MIF a key determinant of inflammation, cardiovascular diseases, and cancer. MIF acts as an upstream regulator within a complex ligand-receptor network comprising the more recently discovered MIF paralog, D-dopachrome tautomerase (D-DT, MIF-2), its cognate receptor CD74, and the chemokine receptors CXCR2, CXCR4, and ACKR3/CXCR7. MIF-driven effects, including leukocyte recruitment, cell migration, and inflammatory signaling, largely depend on the specific MIF receptor repertoire of the involved cell types, which can dynamically change in inflammatory settings. Here, we describe an optimized protocol for the ibidi 3D µ-Slide chemotaxis assay to study MIF-mediated pro-migratory effects on human neutrophils, a prototype of innate immune cells, and CD4+ T cells, a cornerstone of adaptive immunity. Additionally, we discuss the application of current inhibitor strategies to selectively target and identify the receptor pathways involved.