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Kynurenine (KYN) is a non-protein amino acid and major metabolite of tryptophan associated with human health. The acidic form, kynurenic acid (KYNA), has been previously identified in St. John's wort (SJW) tablets and tea, but other metabolites in the pathway are not known in the species. We developed and validated two liquid chromatography-mass spectrometry-based (UHPLC-Orbitrap MS) methods for the quantification of KYNA, KYN, 3-hydroxykynurenine (3-HK), and 3-hydroxyanthranilic acid (3-HAA). We compared two approaches: (1) derivatisation via 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) and (2) analysis of underivatised extracts. KYN was detected in both methods (78% and 101% recovery in underivatised and derivatised, respectively), and 3-HAA was detected only by the underivatised method (95%), while 3-HK was detected only by the derivatised method (90%). Methods were validated for key parameters, including linearity, accuracy, precision, limit of detection (LOD), and limit of quantification (LOQ). Three of the KYN metabolites were detected in <i>in vitro</i>-grown SJW shoots: KYN (101 ng/g), KYNA (104 ng/g), and 3-HAA (670 ng/g). Significantly more KYN and KYNA was detected in greenhouse-grown plants than in tissue-cultured SJW. KYN, KYNA, and 3-HAA were also quantified in commercial preparations of SJW at 0.07 - 0.42 µg/g (KYN), 2.31 - 6.45 µg/g (KYNA), and 0.86 - 3.54 µg/g (3-HAA); 3-HK was not detected in any of our SJW samples. These data support future studies to understand the role of kynurenine metabolites in plant metabolism and plant-based products.