The Klotho gene has been identified as an aging suppressor gene that encodes a transmembrane protein, which is expressed primarily in renal tubules. There are 2 forms of Klotho, membrane and secreted. However, there is a paucity of data on levels of soluble Klotho in diseases like diabetes and kidney disease. We validated an enzyme-linked immunosorbent assay for Klotho and quantitated Klotho levels separately in patients with diabetes and also in patients with chronic kidney disease (CKD). The Klotho assay showed good precision and was linear down to 19 ng/mL. There were no significant effects on Klotho levels with the addition of common interferents such as ascorbate, triglycerides, or hemolysis; only bilirubin (250 mg/L) significantly reduced Klotho levels (P < .05). There was a significant reduction in Klotho levels in samples with glycated hemoglobin (HbA(1c)) levels of 6.5% or more compared with control samples (HbA(1c) < 6.5%; P < .001). We also documented significantly higher levels of Klotho with CKD. Thus, we validated an assay for Klotho and made the novel observation that levels are decreased in diabetes and increased in CKD.