Background: Cervical cancer (CC) is the fourth most prevalent malignancy in women worldwide and a major cause of cancer-related mortality among females in developing countries. P16INK4a is a tumor suppressor protein with an inhibitory role in the proliferation of abnormal cells. The association between the expression of this protein and CC has established it as a diagnostic biomarker for CC.Objectives: This study evaluated the expression level of P16INK4a in cervical squamous cell carcinoma (CSCC). Methods: Eighty-three formalin-fixed paraffin-embedded (FFPE) cervical tissue samples were obtained from the pathology department of Kowsar Gynecology Reference Hospital in Qazvin Province, Iran. The expression level of the P16INK4a gene was investigated using relative quantitative real-time PCR (RT-qPCR). Results: P16INK4a overexpression was observed in 55% (12/22) of cancerous samples, 46% (6/13) of intraepithelial squamous lesions, and 33% (8/24) of tissues with koilocytic changes, with N-fold overexpression of 6.01 (P = 0.001), 3.24 (P = 0.003), and 1.12 (P = 0.007), respectively. Conclusions: The results of this study showed that the expression level of P16INK4a increased with cancer progression. Therefore, the expression level of P16INK4a can be utilized as a biomarker for diagnosis.