State-of-the-art, high-resolution mass spectrometry techniques for acquisition of metabolomic and lipidomic data coupled with advanced computational methods provide new opportunities for interpreting large and complex datasets and comprehending the underlying biological processes of diseases. Both metabolomics and lipidomics strive to obtain comprehensive analyses of small molecules, i.e., metabolites and lipids, in complex biological samples with the goal to identify biomolecular signatures of distinct phenotypes, thus provide potential insights into perturbed pathways and physiological processes and ultimately stages of diseases. This dissertation comprises research studies that collectively describe the development and application of novel mass spectrometry-based metabolomics and lipidomics pipelines for the detection and identification of molecular signatures of perturbations/disease in biological samples. High-resolution mass spectrometry (MS) in combination with ultra-performance liquid chromatography (UPLC), traveling wave ion mobility spectrometry (TWIMS), and computational tools were deployed to facilitate the development of robust and high-throughput analytical approaches. The developed techniques were utilized for the untargeted lipidomic/metabolomic profiling of biological samples derived murine 3T3-L1 cells, mouse hippocampus, and serum of cows.