Target-guided screening of fragments (TGSOF) was developed and employed in the identification of EV-A71 3C protease (3Cpro) inhibitors. We identified 4-acetylpyridine and 3-acetylpyridine as effective P3 fragments of an inhibitor and obtained the corresponding irreversible inhibitors 12c and 12fvia this method. Furthermore, based on 12c and 12f, we have obtained reversible inhibitors 17c and 17f. These results demonstrated that TGSOF is a useful strategy for identifying suitable fragments in developing leads in drug discovery.