Cervical cancer prevention has undergone a major shift with the implementation of human papillomavirus (HPV)-based screening, which offers high sensitivity but limited specificity due to the detection of transient infections. This has created a need for triage methods capable of identifying infections associated with clinically meaningful disease. Primary HPV testing provides a high level of reassurance when negative; however, individuals with a positive result require additional evaluation using triage methods that effectively identify precancerous lesions while avoiding unnecessary colposcopy referrals. This review addresses the role of p16/Ki-67 dual-stain (DS) immunocytochemistry as a biomarker of transforming HPV infection, with a focus on HPV-based screening settings. We discuss its biological basis and diagnostic performance in comparison with cytology, with particular attention to the principles and practical aspects of DS interpretation in routine cytopathological practice. We also summarize current evidence, including data from national studies and routine clinical practice, demonstrating that DS can be effectively integrated into screening pathways and support more informed clinical decision-making. Its increasing use reflects the broader transition toward biologically informed cervical cancer prevention.