Biodiesel is an important low-carbon substitute for petroleum diesel that behaves similarly in combustion engines to petroleum diesel. It has the advantages of being sourced from a wide range of renewable feedstocks, domestic origin, cleaner emission, non-toxicity, and superior lubricating properties. Unfortunately, engine problems related to degradation and deposit formation have resulted from the use of this renewable fuel. These issues are associated with the presence of plant-derived compounds called sterol glucosides [SGs], a sterol is bound to a glucopyranose through an O-ether bond, and their acylated analogues [ASGs]. During the transesterification reaction that produces biodiesel the acyl chain is often liberated from the ASG, yielding an increased concentration of SGs in the product. Under certain conditions SGs can precipitate as a white solid in the biodiesel fuel which can accumulate in filters and critical pipelines in the engines preventing the flow of fuel. Monitoring the levels of these compounds is critical as they directly affect the quality and development of biodiesel. Current analytical techniques for SGs and ASGs in feedstock and biodiesel product involve either gas-liquid chromatography [GC] or High-Pressure Liquid Chromatography [HPLC]. Neither method is straightforward as SGs and ASGs are not volatile and so require derivatisation for GC analysis. They do not absorb light strongly nor exhibit fluorescence and so HPLC detection must be by less sensitive techniques, such as Evaporative Light Scattering. These analyses are also complicated by the complex matrix of the feedstock and biodiesel product and are generally not specific to individual SGs but provide collective quantification. The objective of the present study was to develop an analytical method for the three most commonly reported plant-derived SGs in biodiesel using Liquid Chromatography with Tandem Mass Spectrometry [LC-MS/MS] that is simple, sensitive and specific. The final method involves saponification of the biodiesel, followed by extraction of the SGs to remove matrix effects and their acetylation to yield target compounds that are amenable to LC-MS/MS. A concentration step was necessary to yield highly sensitive results, providing a Method Detection Limit of 10.1 mg L-1 for each SG in biodiesel. This final method was highly specific to the three target compounds although due to the multiple extraction, derivatisation and concentration steps it cannot be claimed to achieve the desired goal of simplicity.