Quantitative amino acid analysis has diverse applications in clinical diagnostics, biomedical research, and agriculture. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) enables more rapid and specific detection of amino acids in comparison to traditional, gold-standard ninhydrin-based methods. However, triple quadrupole mass spectrometers are unable to definitively differentiate isomers and are susceptible to ion suppression, both of which prevent accurate quantitation. Therefore, appropriate chromatography must be applied before ionization.We have shown that two-dimensional LC enables rapid and specific amino acid quantitation without derivatization by resolving isomers, such as alloisoleucine, isoleucine, and leucine, and reducing matrix effects (Le et al., J Chromatogr B Analyt Technol Biomed Life Sci 944:166-174, 2014). In this clinically validated protocol, we provide an updated description of the chromatographic setup and selected reaction monitoring (SRM) transitions. Then, we describe sample processing for serum, plasma, urine, cerebral spinal fluid, and dried blood spots. Most importantly, we outline a singular quantitative design for efficient data analysis of the listed sample types and quality assurance strategies to ensure test fidelity. Lastly, we share extensive knowledge critical to the success of this method. A liquid sample can be processed and be ready for injection within 5 min, and each sample is analyzed by the MS in 14.5 min.