Nicotinamide phosphoribosyl transferase (NAMPT) is a key enzyme in the salvage pathway of mammalian nicotinamide adenine dinucleotide (NAD) biosynthesis, catalyzing the synthesis of nicotinamide mononucleotide from nicotinamide (Nam). The diverse functions of NAD suggest that NAMPT inhibitors are potential drug candidates as anticancer agents, immunomodulators, or other agents. However, difficulty in conducting high-throughput NAMPT assay with good sensitivity has hampered the discovery of novel anti-NAMPT drugs with improved profiles. We combined an in silico screening strategy with a radioisotope (RI)-based enzyme assay and rationally identified promising NAMPT inhibitors with novel structures. AS1604498 was the most potent inhibitor, with an IC₅₀ of 44 n M , and inhibited THP-1 and K562 cell line growth with the IC₅₀ of 198 n M and 673 n M , respectively. The mode of action was found to reduce intracellular NAD following apoptosis, suggesting that these compounds inhibit NAMPT in cell-based assay. This strategy can be used to discover new drug candidates with targets which are difficult to assess through high-throughput screening. Our hit compounds may be used as seed compounds for developing new therapeutics with NAMPT.