SummaryErgosterol (ERG) is a sterol found in fungal cell membranes and is not produced by most plants and animals. Thus, ERG is a potential biomarker of fungal invasion in grain. Previously, we developed an analytical method for ERG in grains by ref uxing samples with methanol-alkali, and performed a single laboratory validation of the method. However, the method is not suitable for screening purpose, which needs to be rapid and simple. Therefore a simplified method was developed for analysis of ERG in grains. In this simplified method, ERG was first extracted with methanol by horizontal shaking for hr. After f ltration, ERG was partitioned into hexane, and then analyzed with reverse phase high performance liquid chromatography (HPLC). Recoveries of ERG obtained with this shaking method were compared with recoveries obtained with the refluxing method. In maize, recovery of ERG at mg/kg using this new shaking method was % of that of the ref uxing method and the relative standard deviation (RSD) of the shaking method and the ref uxing method were . % and %, respectively. In wheat, recovery of ERG at mg/kg by the shaking method was % of that of the ref uxing method, and the RSD of the shaking method and the ref uxing method were . % and . %, respectively. This study demonstrates this simplif ed protocol for ERG analysis in grains to be a suitable method for screening purposes.