Abstract The main step during biodiesel production is the catalytic transesterification of triglycerides. Glycerol and fatty acids are by-products of the biodiesel production and considered as contaminants in the final product. By selectively measuring the amount of fatty acids and glycerol with different substitution levels, the yield of this step and the quality of the final biodiesel can be determined. This study examines the application of phosphitylation of glycerol hydroxyl groups with 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane followed by 31P NMR analysis to provide a rapid quantitative analytical technique for the determination of substitution patterns on partially esterified glycerols, alcohols and the detection of fatty acids. 31P NMR chemical shift data was established with a series mono and di-substituted esters of glycerol, fatty acids and alcohols, then utilized to characterize commercial glycerol and biodiesel samples.