This Ph.D. thesis explores the development of multidimensional mass spectrometry-ion mobility-based methods and separation science workflows for applications in metabolomics, forensics, pharmaceuticals, and food sciences. The first chapter introduces the separation techniques, Liquid Chromatography (LC), Supercritical Fluid Chromatography (SFC), and their hyphenation to Mass Spectrometry (MS) and Ion Mobility Spectrometry (IMS) for multidimensional separation. The second chapter describes an automated parallel derivatization workflow for qualitative and quantitative analysis using LC-SWATH-MS and was applied to quantify 40 metabolites in urine samples from a human prostate cancer study. The third chapter, divided into four sections, is dedicated to the implementation, characterization, and development of analytical methods of vacuum Differential Mobility Spectrometry (vDMS) coupled to a triple quadrupole mass analyzer. The fourth chapter describes a metabolomic study for pediatric Chronic Kidney Disease (CKD) using an integrated workflow for the characterization of xenobiotics and endogenous metabolites in urine and plasma samples.