Differentiation of enantiomers represents an important research area for pharmaceutical, chemical, and food industries. However, enantiomer separation is a laborious task that demands complex analytical techniques, specialized equipment, and expert personnel. In this respect, discrimination and quantification of d- and l-α-amino acids is no exception, generally requiring extensive sample manipulation, including isolation, functionalization, and chiral separation. This complex sample treatment results in high time costs and potential biases in the quantitative determination. Here, we present an approach based on the combination of non-hydrogenative parahydrogen-induced hyperpolarization and nuclear magnetic resonance that allows detection, discrimination, and quantification of d- and l-α-amino acids in complex mixtures such as biofluids and food extracts down to submicromolar concentrations. Importantly, this method can be directly applied to the system under investigation without any prior isolation, fractionation, or functionalization step.