We have developed a surface plasmon resonance (SPR)-based inhibition in solution assay (ISA) to search for inhibitors of the medium affinity (KD = 0.8 μM) interaction between an E6-derived peptide (E6peptide) immobilized on the sensor and a PDZ domain (MAGI-1 PDZ1) in the mobile phase. DZ domains are widespread protein-protein interaction modules that recognize the C-terminus of various partners. Simulations indicated that relatively low compound concentrations (10 μM) and limited peptide densities (Rmax < 200 resonance units) should allow the detection of inhibitors with a target affinity close to 100 μM, which was then demonstrated experimentally. ISA screening, carried out on the Prestwick Chemical Library® (1120 compounds), identified 36 compounds that inhibited the interaction by more than 5%. Concentration-dependent ISA, carried out on a subset of 19 potential inhibitors, indicated that 13 of these indeed affected the interaction between MAGI-1 PDZ1 and the E6peptide. No effect was observed for 84 compounds randomly chosen among noninhibitors. One of the four best inhibitors was a peptide binder, and three were PDZ binders with KD in the 10-50 μM range. We propose that a medium (μM) affinity between the target and surface-bound partner is optimal for SPR-based ISA screening.