The factor-inhibiting hypoxia-inducible factor (FIH) hydroxylates the asparagine 803 (Asn803) residue of the hypoxia-inducible factor 1alpha (HIF-1alpha), and the modification abrogates the transcriptional activity of HIF-1alpha. Because FIH is more active on HIF-1alpha than prolyl hydroxylase domain proteins under hypoxic conditions, its inhibitors have potential to be developed as anti-ischemic drugs targeting normal cells stressed by hypoxia. In this study, the authors developed the first monoclonal antibody, SHN-HIF1alpha, specifically to Asn803 hydroxylated HIF-1alpha and a sensitive assay system for FIH inhibitors using the monoclonal antibody (Mab). SHN-HIF1alpha showed 740 times higher affinity to the Asn803 hydroxylated HIF-1alpha peptide than the unmodified one. An enzyme-linked immunosorbent assay-based system using SHN-HIF1alpha displayed at least 30 times more sensitivity than previous methods for screening FIH inhibitors and was easily applicable to develop a high-throughput screening system. SHN-HIF1alpha also showed an Asn803 hydroxylation-dependent specificity to HIF-1alpha in cells. Taken together, the results suggest that it may be used to analyze the in vivo and in vitro activities of FIH inhibitors.