The available therapeutic approaches for cervical cancer can seriously affect the fertility potential of patient; thus, there is a pressing requirement for less toxic and targeted therapies. The membrane proteome is a potential source of therapeutic targets; however, despite the significance of membrane proteins in cancer, proteomic analysis has been a challenging task due to their unique biochemical properties. The aim of the present study was to develop an efficient membrane protein enrichment protocol, and to the best of our knowledge, to compare for the first time the expression pattern of membrane proteins of one normal cell line, HCK1T, and three cervical cancer cell lines, C33A, a human papilloma virus (HPV)-negative cell line, and two HPV-positive cell lines, SiHa (HPV16+) and HeLa (HPV18+). The study aimed to identify the proteins that are involved in cervical carcinogenesis and may constitute novel drug targets. Membrane protein isolation, liquid chromatography coupled with tandem mass spectrometry proteomics and bioinformatics analysis were performed in the membrane fraction of the informative cervical cell lines following a novel enrichment protocol. The percentages of membrane and transmembrane proteins in the enrichment protocol were higher compared with those of the corresponding data derived from total cell extract analysis. Differentially expressed proteins were detected by the comparison of the cervical cancer cell lines with the normal cell line. These proteins constitute molecular features of cancer pathology and participate in biological pathways relevant to malignancy, including 'HIPPO signaling', 'PI3K/Akt signaling', 'cell cycle: G2/M DNA damage checkpoint regulation' and 'EIF2 signaling'. These unique membrane protein identifications offer insights on a previously inaccessible region of the cervical cancer proteome, and may represent putative diagnostic and prognostic markers, and eventually therapeutic targets.