Background: IDO-1 catalyzes the degradation of trp to kyn and can promote tumor escape from host immunosurveillance. IDO-1 expression evaluation has potential importance in identification of a population with worse prognosis and is important in the clinical evaluation of IDO-1 inhibitors. We have developed an optimized assay for IDO-1 specific message detection using RNAscope technology (RST) and applied it to an analysis of human melanoma (MEL) samples. Methods: 25 MEL samples were evaluated for IDO-1 by IHC and the samples used to optimize the RST assay. IDO-1 expression could be demonstrated in tumor cells (TC) as well as in immune cells (IC) infiltrating the tumor and in IC in the peritumoral area. A scoring method was developed to classify IDO-1 expression in all three populations. An H-score of 5 or greater was used as an arbitrary cutoff for IDO-1 positivity. An additional 27 MEL samples, collected as part of the ECHO202/KN037 study of epacadostat in combination with pembrolizumab, were evaluated. Results: IHC for IDO-1 showed 24% positive samples for tumor expression. As expected based on enhanced sensitivity, RST identified 56% as positive – 83% of the samples were positive by both assays. With IHC, 60% were positive for intratumoral IC expression of IDO-1 while RST identified 68% as positive for IC expression – 93% of the samples were positive by both methods. Analysis of the samples from the ECHO 202 study showed that 85% were positive for TC IDO-1 expression, while 59% were positive for IC IDO-1 expression with 52% positive for both. Of 22 PD-L1+ samples, 20 were positive for IDO-1 in either TC or IC. Of PD-L1- samples, 4 were positive for IDO-1 in either TC or IC. There was a trend toward higher IDO-1 expression in PD-L1+ tumors. In this data set there was no association of IDO-1 expression with BRAF mutation status, elevated LDH or prior exposure to immune therapies. Conclusions: RST detection has the potential to provide a robust and quantitative assay to evaluate IDO1 expression in TC and IC. Further refinement of this assay and determination of its utility in assessing predictors of patient response in MEL patients undergoing combination therapy with epacadostat and pembrolizumab will take place in ECHO-301/KN252 (NCT02752074).