In this study, we employ a Taguchi L'9 orthogonal array design to optimize a sequential non-polar and polar extraction of the model organism C. elegans for untargeted reversed-phase (RP) and hydrophilic interaction liquid chromatography (HILIC) LC-MS metabolomics, respectively to provide coverage of the lipidome (RP) and polar metabolome (HILIC) from the same sample homogenate. Optimized conditions were validated using NMR as an orthogonal and quantitative analytical method. The results demonstrate the benefits of the Taguchi DOE approach in its application to the field of untargeted metabolomics and highlights the capabilities of simultaneously optimizing the base conditions for a sequential extraction. Including such a strategy during the experimental design phase enhances the experimental throughput and overall metabolome coverage for metabolomics studies while informing the investigator of the advantages and limitations of their specific extraction method and sample preparation approach.