In this study, we demonstrated the efficiency and applicability of a Taguchi DOE approach to optimize a sequential non-polar and polar extraction of the model organism C. elegans for analysis by LC-MS and NMR, two orthogonal technologies at the forefront of the metabolomics field. Such a strategy will also be beneficial to investigators exploring other model or emerging nonmodel organisms. To our knowledge, this is the first application of the Taguchi method to an untargeted metabolomics extraction method for both mass spectrometry and nuclear magnetic resonance. Selected sample preparation parameters for both RP and HILIC chromatography were optimized simultaneously. This methodology significantly decreased the number of samples necessary for optimization. A conventional OFAT approach would not only require nearly 10x this number of samples, but also limit our ability to determine any interactions between the individual factors. While we have showcased the application of this approach to an untargeted workflow, there is immense potential for applications to other biochemical and metabolomics studies such as targeted workflows to improve quantitation, additional biological matrices, and metabolite derivatization. The Taguchi DOE method is a highly adaptable and scalable DOE approach that enables the optimization of multiple extraction parameters and investigation of the interactions between factors, all while decreasing manual labor, consumables costs, and analysis time to further enable high throughput science.