Currently, there is neither a published ELISA method nor it is clear whether chemiluminescence substrates would provide better sensitivity vs. colorimetric substrates for measuring human IL-23-a recently described Type-1 immunity associated cytokine. Initially, we optimized a colorimetric ELISA using p-nitro-phenyl phosphate substrate. Subsequently, we compared it with chemiluminescence substrates that provided approximately 5-fold enhanced sensitivity (mean sensitivity; 26.3 pg/mL vs. colorimetric assay, 131 pg/mL; p < 0.01). Both methods were reliable, with <10% inter- and intra-assay variations. We then found that the chemiluminescence method was useful in situations where human IL-23 was not readily measurable by a colorimetric method.