There is evidence that testing for human papillomavirus (HPV) E6/E7 mRNA is more specific than testing for HPV DNA. A retrospective study was carried out to evaluate the performance of the PreTect HPV-Proofer E6/E7 mRNA assay (Norchip) as a triage test for cytology and HPV DNA testing. This study analyzed 1,201 women, 688 of whom had a colposcopy follow-up and 195 of whom had histology-confirmed high-grade intraepithelial neoplasia or worse (CIN2؉). The proportion of positive results and the sensitivity and specificity for CIN2؉ were determined for HPV mRNA in comparison to HPV DNA and cytology. All data were adjusted for follow-up completeness. Stratified by cytological grades, the HPV mRNA sensitivity was 83% (95% confidence interval [CI] ؍ 63 to 94%) in ASC-US (atypical squamous cells of undetermined significance), 62% (95% CI ؍ 47 to 75%) in L-SIL (low-grade squamous intraepithelial lesion), and 67% (95% CI ؍ 57 to 76%) in H-SIL (high-grade squamous intraepithelial lesion). The corresponding figures were 99, 91, and 96%, respectively, for HPV DNA. The specificities were 82, 76, and 45%, respectively, for HPV mRNA and 29, 13, and 4%, respectively, for HPV DNA. Used as a triage test for ASC-US and L-SIL, mRNA reduced colposcopies by 79% (95% CI ؍ 74 to 83%) and 69% (95% CI ؍ 65 to 74%), respectively, while HPV DNA reduced colposcopies by 38% (95% CI ؍ 32 to 44%) and by 15% (95% CI ؍ 12 to 19%), respectively. As a HPV DNA positivity triage test, mRNA reduced colposcopies by 63% (95% CI ؍ 60 to 66%), having 68% sensitivity (95% CI ؍ 61 to 75%), whereas cytology at the ASC-US؉ threshold reduced colposcopies by 23% (95% CI ؍ 20 to 26%), showing 92% sensitivity (95% CI ؍ 87 to 95%). In conclusion, PreTect HPV-Proofer mRNA can serve as a better triage test than HPV DNA to reduce colposcopy referral in both ASC-US and L-SIL. It is also more efficient than cytology for the triage of HPV DNA-positive women. Nevertheless, its low sensitivity demands a strict follow-up of HPV DNA positive-mRNA negative cases.