The development of a validated method, applicable for the measurement of tryptophan (TRP) and serotonin (5-HT), and that of the neuroprotective branch of the kynurenine pathway from several different biological matrices, including mouse brain, is described. Following the spectral analysis of the metabolites, they were quantified with reversed-phase high-performance liquid chromatography (HPLC), using separate internal standards (ISs) for UV (3-nitro-L-tyrosine) and fluorescent (the newly utilized 4-hydroxyquinazoline-2-carboxylic acid) detectors. With regard to validation parameters, selectivity, linearity, limit of detection, limit of quantification, precision and recovery were determined. Although the linearity ranges were different for the assessed matrices, the correlation coefficient was >0.999 in each case. Furthermore, good intra- and inter-day precision values were obtained with coefficient of variation <5%, and bias <6.5% (except the 5-HT level in brain samples), respectively. The recoveries varied between 82.5% and 116%. The currently developed methods yield opportunities for the assessment of concentration changes in the TRP metabolism from a wide range of biological matrices, therefore they may well be utilized in future clinical and preclinical studies, especially in view that so many metabolites with the application of ISs have not been detected from mouse brain with such a simple HPLC method before.