The absence of Klotho (KL) from mice causes the development of disorders associated with human aging and decreased longevity, whereas increased expression prolongs lifespan. With age, KL protein levels decrease, and keeping levels consistent may promote healthier aging and be disease-modifying. Using the KL promoter to drive expression of luciferase, we conducted a high-throughput screen to identify compounds that activate KL transcription. Hits were identified as compounds that elevated luciferase expression at least 30%. Following validation for dose-dependent activation and lack of cytotoxicity, hit compounds were evaluated further in vitro by incubation with opossum kidney and Z310 rat choroid plexus cells, which express KL endogenously. All compounds elevated KL protein compared with control. To determine whether increased protein resulted in an in vitro functional change, we assayed FGF23 (fibroblast growth factor 23) signalling. Compounds G-I augmented ERK (extracellular-signal-regulated kinase) phosphorylation in FGFR (fibroblast growth factor receptor)-transfected cells, whereas co-transfection with KL siRNA (small interfering RNA) blocked the effect. These compounds will be useful tools to allow insight into the mechanisms of KL regulation. Further optimization will provide pharmacological tools for in vivo studies of KL.