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It is urgent to develop an accurate approach to improve the predictive performance of hrHPV-based screening. The aim is to evaluate the performance of p16/Ki-67 and p16/MCM2 staining to triage high-risk human papillomavirus- (hrHPV-) positive women. Cervical specimens were collected from eligible women and tested for hrHPV genotyping, cytology, p16/Ki-67, and p16/MCM2 staining at baseline. Women were invited to participate in follow-up screening by cytology and hrHPV testing at 24 months. Positive women received colposcopy and biopsies. Histopathological diagnoses were the gold standard. 485 women came back for the follow-up screening. The positive rate of p16/Ki-67 was 20.2% and of p16/MCM2 was 27.2%. The positive rates of p16/Ki-67 (  P  < 0.001) and p16/MCM2 ( P =0.021) were increased by the severity of histopathology findings. Among hrHPV-positive women, the sensitivity, specificity, PPV, and NPV for p16/Ki-67 were 90.9%, 67.0%, 16.5%, and 99.0%, and for p16/MCM2 were 81.8%, 43.1%, 9.4%, and 97.1%. The sensitivity of cytology for triaging hrHPV-positive women were lower than p16/Ki-67 ( P =0.012) and p16/MCM2 ( P =0.065). The cocktail staining did not add sensitivity to p16/Ki-67 or p16/MCM2 staining alone ( P  > 0.05), however, cutting down the specificity of p16/Ki-67 staining alone with statistical significance (67.0% vs. 40.2%,  P  < 0.001). The risk of CIN2+ within 24 months for hrHPV-positive but triaging negative women at baseline was 0.5 (0.1–2.7), 0.7 (0.1–4.1), and 2.4 (1.1–5.0) for p16/Ki-67, p16/MCM2, and cytology, respectively. As an objective and accurate immunocytochemical staining, the p16/Ki-67 and p16/MCM2 dual staining performed better than cytology to triage positive hrHPV. On condition that high-quality cytology is unavailable, immunocytochemical staining by p16/Ki-67 or p16/MCM2 is an option for triaging hrHPV-positive women. The combination of p16/Ki-67 and p16/MCM2 could not improve the accuracy in detecting CIN2+.

The Performance of Immunocytochemistry Staining as Triaging Tests for High-Risk HPV-Positive Women: A 24-Month Prospective Study