The cytokine MIF is implicated in several autoimmune and inflammatory conditions and is elevated in several types of cancer, making it a promising therapeutic target. Small-molecule MIF inhibitors provide tools to elucidate MIFs’ immune regulatory roles, and their further optimization could lead to potential therapeutics. Surface plasmon resonance (SPR) is a relatively high-throughput and sensitive biophysical technique used in drug discovery for screening small organic compounds (termed fragments) for their interactions with macromolecules. SPR analysis can provide the association and dissociation rate constants and the equilibrium binding constant for protein interactions. As such, SPR also provides a valuable orthogonal technique to determine compound binding (KA and KD) and correlate it with inhibitory activity (IC50) observed in functional assays or cell culture experiments. This helps researchers discriminate false positive results from off-target effects or compound misbehavior. Establishing SPR conditions required to immobilize active and stable proteins for analysis can be challenging. Hence, we present a robust SPR assay for MIF, outlining basic approaches and tips for screening and validating fragments.