In this study, we present a rapid, robust and sensitive method for quantification of plant amino acid uptake using universally (U) (13C, 15N)-labelled amino acids and gas chromatography-mass spectrometry (GC-MS). Amino acids were analysed as their tert-butyldimethylsilyl (tBDMS) derivatives and displayed detection limits in the range 10–100 fmol on column, depending on the amino acid. The technique allows for simultaneous detection and quantification of both unlabelled and isotopically labelled species of amino acids. This makes simple quantification of plant amino acid uptake from an isotopically labelled source possible. The analytical variation was low, concerning total amino acid concentrations (relative standard deviation, rsd, less than 5.3%) as well as enrichment of U-13C, 15N-labelled glycine (Gly), arginine (Arg) and glutamic acid (Glu) (rsd<2.1%). An application of the GC-MS method was conducted on non-mycorrhizal Pinus sylvestris roots supplied with U-13C, 15N-labelled amino acids. Intact, labelled amino acids were traced in root extracts. This provided conclusive evidence of plant root uptake of intact amino acids. Uptake rates of the three amino acids Gly, Glu and Arg in the range 0.5–37.9 μmol g−1 dry weight h−1 were recorded. These rates are comparable with those recorded in earlier studies of amino acid uptake, using other methods, as well as uptake rates measured for nitrate and ammonium.