Biofuels continue to be of key importance to the energy sector as more renewable fuel solutions are developed to contribute to global climate goals and resolve energy shortages. Fatty Acid Methyl Ester (FAME) Biodiesel (B100) is a key component for blending with fossil-fuel diesel in portions of up to 30% depending on the region and engine type. Standards specifications for B100 biodiesel exist in the form of ASTM D6751 – Standard Specification for Biodiesel Fuel Blend Stock (B100) for Middle Distillate Fuels and EN14214 – fatty acid methyl esters (FAME) for use in diesel engines and heating applications – requirements and test methods. For both standards the specifications state that methanol content in B100 should not exceed 0.2 % m/m. Methanol is an important requirement in the transesterification reaction used to produce the FAME. The methanol is separated after the completion of the reaction and precise analysis is required to ensure that any residual methanol content within the FAME is within specification to ensure it is fit for purpose. The standard test method to determine methanol content in B100 is EN14110 which specifies the use of manual or automatic headspace gas chromatography. This application note covers the analysis of methanol in B100 utilizing the PerkinElmer GC 2400™ System and PerkinElmer HS 2400™ Headspace Sampler.