The development and validation of an analytical method for the simultaneous analysis of five neutral lipids in Trypanosoma cruzi epimastigotes by GC-MS is presented in this study. The validated method meets all validation parameters for all components and the chromatographic conditions have been optimized during its development. This analytical method has demonstrated good selectivity, accuracy, within-day precision, recovery and linearity in each of the established ranges. In addition, detection and quantification limits for squalene, cholesterol, ergosterol and lanosterol have been improved and it is worth highlighting the fact that this is the first time that squalene-2,3-epoxide validation data have been reported. The new validated method has been applied to epimastigotes treated with compounds with in vitro anti-T.cruzi activity. This new methodology is straightforward and constitutes a tool for screening possible sterol biosynthesis pathway inhibitors in Trypanosoma cruzi, one of the most studied targets in Chagas disease treatment. Therefore, it is an interesting and useful contribution to medicinal chemistry research.