Tryptophan and its metabolites are of great interest in understanding the pathogenesis of multiple sclerosis (MS). The total levels of tryptophan and its metabolites, kynurenine and kynurenic acid were determined in plasma by capillary liquid chromatography electrospray ionisation tandem mass spectrometry. This is the first report of the plasma levels of these analytes in healthy controls and relapsing-remitting MS patients receiving long-term and acute interferon-beta (IFN-beta) treatment. Twenty-four hours post-administration increased kynurenine levels (first IFN MS versus healthy, P = 0.042) and kynurenine/tryptophan ratio (K/T; first IFN MS versus healthy, P =0.027; first IFN MS versus long-term IFN MS, P = 0.036) were found. The long-term IFN MS group had higher K/T ratios at 4 and 12 h post-administration (P = 0.015 and 0.009, respectively). The increase of K/T ratio in the first IFN MS group indicate an induction of the enzyme indolamine-2,3-dioxygenase (IDO), as reported earlier in experimental allergic encephalomyelitis. As IDO is participating in both inflammatory and neurodegenerative processes, further knowledge of its involvement in the pathogenesis of MS is of great importance.