Expression of high-risk HPV oncogenes results in a strong overexpression of cellular protein p16(INK4a). Immunohistochemical staining for p16(INK4a) is widely used as diagnostic marker. However, p16(INK4a) upregulation was also described as a biomarker of age. Here we analyzed p16(INK4a) expression in cervical smears to investigate if patient age may influence p16(INK4a)-based cervical cancer diagnosis. p14(ARF) was analyzed as a related supportive biomarker. Cervical scrapes were taken and stored in RNAlater. Total RNA was extracted, and cDNA was analyzed for expression of p16(INK4a) and p14(ARF) relative to β-actin, by real-time reverse transcriptase PCR SYBR-Green I assays. Patient-derived smears referred as HSIL (n=45) had 6.27-fold higher p16(INK4a) mRNA expression than smears of cytologically normal and HPV-negative persons (n=48). Expression of p14(ARF) was 4.87-fold higher. When women with normal diagnoses were stratified for age, a significantly enhanced p16(INK4a) (2.88-fold) and p14(ARF) (1.9-fold) expression was observed as a consequence of ageing. A significant age-dependent upregulation was also observed in older HSIL patients (2.54-fold). Our study revealed significantly enhanced expression of p16(INK4a)/p14(ARF) mRNA in cervical scrapes referred to as HSIL compared with normal women. An age-dependent bias has to be considered when quantifying these tumor suppressor genes, with respect to cervical cancer development.