A quantitative method is reported for the estimation of glycerol during transesterification of oil to form biodiesel. The reagent used to derivatize glycerol was 9,9-dimethoxyfluorene. Glycerol is estimated by both UV-visible spectrophotometric and high performance liquid chromatography methods. Using the former method, detection limits of 0.05% w/w of glycerol in biodiesel was established. Validation of the developed method was done using the Greenhill method for determination of free glycerol formed during the transesterification reaction. © 2010 AOCS.