Screening combinatorial libraries for inhibition of Paxillin binding to the cytoplasmic tail of the integrin α4 provided the first inhibitors of this protein-protein interaction implicated in enhanced rates of cell migration and chronic inflammation. The preparation of substructure analogs of the lead identified features required for activity, those available for modification, and those that may be removed. The most potent lead structure was shown to inhibit α₄β₁-mediated human Jurkat T cell migration in a dose-dependent manner, validating the intracellular Paxillin/α4 interaction as a useful and unique target for therapeutic intervention. Moreover, the lead structure emerged from a library that was prepared in two formats: (1) a traditional small mixture format composed of 100 mixtures of 10 compounds and (2) a positional scanning library. Their parallel testing provided the rare opportunity to critically compare two approaches.