Chlorpyrifos is a neurotoxic insecticide that is widely used in the agricultural sector of Ghana. The main objective of this study was to evaluate the levels of chlorpyrifos exposure and health risk among applicators (n = 21) on irrigated rice farms in Ghana, based on a typical application event. Pre- and post-application urine samples (24-h) were collected from the applicators and analysed for 3,5,6-trichloro-2-pyridinol (TCP), using LC-MS/MS. The levels of chlorpyrifos-absorbed dose with the applicators were estimated from the urinary TCP levels. Prior to application, the median absorbed dose of chlorpyrifos (background exposure) with the applicators was 0.2 μg/kg/day (range 0.05 to 2 μg/kg/day). Following application, the median absorbed dose of chlorpyrifos (application exposure) increased 30-fold to 6 μg/kg/day (range 0.7 to 74 μg/kg/day). The mean elimination halflife (t1/2) of chlorpyrifos was calculated to be 50 h. Hazard quotient (HQ) values (HQ > 1) obtained with the chronic (10 μg/kg/ day) and acute (100 μg/kg/day) guideline values of the WHO suggested no risk of chronic or acute health effects, respectively, among both the median and 5% highly exposed groups. However, HQ values (HQ > 1) obtained with the chronic (0.3 μg/kg/day) and acute (5 μg/kg/day) guideline values of the USEPA suggested risk of chronic and acute health effects, respectively, among both the median and 5% highly exposed groups. The quantity of chlorpyrifos formulation applied, spraying duration, and the number of spray tanks applied significantly correlated with the absorbed dose levels of chlorpyrifos from application exposure. Therefore, these factors suggest means to reduce exposure and consequent health risk among the applicators.