The accurate detection and genotyping of high-risk human papillomavirus (HR-HPV) are critical for cervical cancer screening and epidemiological investigations. GeneFirst Papilloplex® HR-HPV is a new CE-IVD-marked real-time PCR test based on patented multiplex probe amplification technology. Papilloplex® HR-HPV provides the simultaneous detection and differentiation of 14 HR-HPV genotypes (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68a/b) in a single closed-tube reaction ensuring rapid, cost-effective, and contamination-free results. In this study, the analytical performance characteristics in terms of the assay's sensitivity, specificity, range, reproducibility, and cross-reactivity were evaluated. Papilloplex® HR-HPV provided sensitive detection and differentiation of 14 HR-HPV types with highly reproducible results. The differential HR-HPV specificity and sensitivity were further confirmed through the participation in the WHO HPV Laboratory Network Proficiency Study (2014). Overall, GeneFirst Papilloplex® HR-HPV assay demonstrated a robust analytical performance with reproducible and reliable results in the detection of HR-HPV genotypes.