Introduction: Human papillomavirus (HPV) is a small, non-enveloped double-stranded circular DNA virus. The high risk types of HPV are claimed to be responsible for over 99% of cervical cancers while the most common oncogenic genotypes 16 and 18, implicated in approximately 70% of all cervical cancers. HPV has six early genes (E1, E2, E4, E5, E6 and E7) that code for regulatory proteins involved in viral DNA replication, transcription control and cellular transformation. The E7 protein is responsible for the escape from cell cycle arrest in HPV infected cells by binding to the retinoblastoma protein (pRB) through its LXCXE binding motif. This inhibits the tumour suppressor protein from regulating the cell cycle progression from G1 to S phase. In this study, the pocket domain of pRB which is targeted by LXCXE is used as a target to design peptide inhibitors using in silico methods. Methods: Biovia Discovery Studio Visualizer and UCSF Chimera softwares were used in designing the peptides. Results: Two crystal structures: 1GUX and 4YOZ were superimposed and studied. The similar amino acid sequences which bind to LXCXE were cut to form the potential peptide inhibitors. Based on this, peptide 1 was selected for further in vitro analysis. The cytotoxicity of the peptide 1 was analysed on three cell lines: CaSki (HPV16+ cervical cancer cell), C33a (cervical cancer cell), and HaCaT (normal keratinocyte). As a result, the IC50 of one of the designed peptide inhibitor – peptide 1, on CaSki cell line was 180 μM. The inhibitory effect of the peptide 1 was also analysed validated using Western Blot using antibodies from the B-Myb and pRB family. The changes in cell cycle before and after treatment of the peptide inhibitor will be further monitored using a cell analyser. Conclusion: Peptide 1 had shown potential as an inhibitor of the HPV E7 protein based on the in silico analysis, but further functional studies are needed to validate its potential.