AbstractThe dim light melatonin onset (DLMO) is the current gold standard biomarker of the timing of the central circadian clock in humans and is often assessed from saliva samples. To date, only one commercially available salivary melatonin assay is considered accurate at the low daytime levels required to accurately detect the DLMO (Novolytix RIA RK‐DSM2). The aim of this study was to conduct the first independent evaluation of a newly improved enzyme‐linked immunosorbent assay (ELISA; Novolytix MLTN‐96) and compare it with the recommended radioimmunoassay (RIA)—both in terms of melatonin concentrations and derived DLMOs. Twenty participants (15 females, 18–59 years old) provided saliva samples every 30 min in dim light starting 6 h before their habitual bedtime, yielding a total of 260 saliva samples. Both the RIA and ELISA yielded daytime melatonin concentrations <2 pg/mL, indicating adequate accuracy to detect the DLMO. The melatonin concentrations from the two assays were highly correlated (r = .94, p < .001), although the RIA yielded lower levels of melatonin concentration than the ELISA, on average by 0.70 pg/mL (p = .006). Seventeen DLMOs were calculated from the melatonin profiles and the DLMOs from both assays were not statistically different (p = .36) and were highly correlated (r = .97, p < .001). Two DLMOs derived from the RIA occurred more than 30 min earlier than the DLMO derived from the ELISA. These results indicate that the new Novolytix ELISA is an appropriate assay to use if the Novolytix RIA is not feasible or available.