Inhibitors of indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO) are potential drugs for the treatment of tumor and neurological diseases. A variety of bioassays have been developed to evaluate IDO1/TDO (IDO1 and/or TDO) inhibitors, with uncertainty regarding how the differences in the assay methods or protocols may influence the assay outcomes. The enzymatic assays of IDO1/TDO are usually performed with NFK assay and Kyn adduct assay while the cellular assays of IDO1 are carried out with Hela assay and HEK293 assay. The present study focused on the comparison of the most common bioassays of IDO1/TDO. In addition, the effects of major factors of bioassays such as reaction time and culture medium on the assay outcomes were evaluated. The study will provide reference for the researchers to select IDO1/TDO inhibitors with bioassays, and promote the development of IDO1/TDO inhibitors.