The cofactor nicotinamide adenine dinucleotide (NAD⁺) plays a key role in a wide range of physiological processes and maintaining or enhancing NAD⁺ levels is an established approach to enhancing healthy aging. Recently, several classes of nicotinamide phosphoribosyl transferase (NAMPT) activators have been shown to increase NAD⁺ levels in vitro and in vivo and to demonstrate beneficial effects in animal models. The best validated of these compounds are structurally related to known urea-type NAMPT inhibitors, however the basis for the switch from inhibitory activity to activation is not well understood. Here we report an evaluation of the structure activity relationships of NAMPT activators by designing, synthesising and testing compounds from other NAMPT ligand chemotypes and mimetics of putative phosphoribosylated adducts of known activators. The results of these studies led us to hypothesise that these activators act via a through-water interaction in the NAMPT active site, resulting in the design of the first known urea-class NAMPT activator that does not utilise a pyridine-like warhead, which shows similar or greater activity as a NAMPT activator in biochemical and cellular assays relative to known analogues.