Indoleamine 2,3-dioxygenase 1 (IDO1) catalyzes the first and rate-limiting reaction of L-tryptophan (Trp) conversion into L-kynurenine (Kyn). The depletion of Trp, and the accumulation of Kyn have been proposed as mechanisms that contribute to the suppression of the immune response—primarily evidenced by in vitro study. IDO1 is therefore considered to be an immunosuppressive modulator and quantification of IDO1 metabolism may be critical to understanding its role in select immunopathologies, including autoimmune- and oncological-conditions, as well as for determining the potency of IDO1 enzyme inhibitors. Because tryptophan 2,3-dioxygenase (TDO), and to a significantly lesser extent, IDO2, also catabolize Trp into Kyn, it's important to differentiate the contribution of each enzyme to Trp catabolism and Kyn generation. Moreover, a great variety of detection methods have been developed for the quantification of Trp metabolites, but choosing the suitable protocol remains challenging. Here, we review the differential expression of IDO1/TDO/IDO2 in normal and malignant tissues, followed by a comprehensive analysis of methodologies for quantifying Trp and Kyn in vitro and in vivo, with an emphasis on the advantages/disadvantages for each application.