Targeted metabolomics and lipidomics are increasingly utilized in clinical research, providing quantitative and comprehensive assessments of metabolic profiles that underlie physiological and pathological mechanisms. These approaches enable the identification of critical metabolites and metabolic alterations essential for accurate diagnosis and precision treatment. Mass spectrometry, in combination with various separation techniques, offers a highly sensitive and specific platform for implementing targeted metabolomics and lipidomics in clinical settings. Nevertheless, challenges persist in areas such as sample collection, quantification, quality control, and data interpretation. This review summarizes recent advances in targeted metabolomics and lipidomics, emphasizing their applications in clinical research. Advancements, including microsampling, dynamic multiple reaction monitoring, and integration of ion mobility mass spectrometry, are highlighted. Additionally, the review discusses the critical importance of data standardization and harmonization for successful clinical implementation.