In this study we report on the specificity profiling of the MAP kinase inhibitors 1, 2, and 3 in a panel of 78 protein kinases including the MAPK isoforms p38(a,b,c,d), JNK1/2/3, and ERK1/2/8 showing 3-(4-fluorophenyl)-4-pyridin-4-ylquinolin-2(1H)-one (1) to be highly selective for p38aMAPK with an IC50 of 1.8 lM. In contrast, besides p38a the isoxazoles 2 and 3 significantly inhibited JNK2/3 and further kinases beyond the MAPK family such as PKA, PKD, Lck, and CK1. By using sequence alignment and homology models of different members of the MAPK family the binding mode determining selectivity of 1 for the p38a isoform was investigated. For lead optimization of 1 a straightforward tandem-Buchwald-aldol synthetic approach toward the flexible decoration of the quinolin-2(1H)-one scaffold was employed. SAR for derivatives of 1 at the isolated p38aMAPK are presented.