Cervical squamous cell carcinoma (SCC) is among the most common malignancies in women worldwide. In developed countries routine cytology screening has dramatically reduced SCC mortality within the last three decades. High risk (HR) human papilloma virus (HPV) infection is the main causal factor in nearly 100% of invasive SCCs, in most cases of low grade squamous intraepithelial lesion (LSIL) and in nearly all cases of high grade squamous intraepithelial lesion (HSIL). Detection of HR-HPV DNA has been extensively evaluated for the triage of patients with low grade cytological abnormalities in order to identify those at greatest risk for underlying or developing HSIL or SCC. However, the vast majority of HR-HPV-positive precursor lesions will not progress to invasive cancer. A variety of other screening tools are available which aim to stratify clinically significant HPV infections and cytological alterations. Matrix assisted laser desorption/ionization (MALDI) imaging mass spectrometry is a promising technology to assist in this endeavor. It delivers accurate mass spectrometric information of the sample's proteins and enables the visualization of the spatial distribution of protein expression profiles in correlation with histological features. In this study, 18 samples with Pap IIID or higher (>LSIL) and 14 samples with Pap I-II (WNL) were analyzed by MALDI imaging mass spectrometry (IMS). A genetic algorithm was applied to classify spectra resulting in an overall cross validation, sensitivity for Pap IIID and Pap I-II of 83.7%, 88.9% and 78.6%, respectively. As this IMS based approach allows for unbiased and automated classifiction of cytological samples it appears to be a promising tool for stratification of cervical Pap smears.