A shift from conventional cytology to a molecular approach could improve cervical cancer screening. This proof-of-concept study aims to develop a high-content imaging platform for the simultaneous detection of multiple biomarkers for cervical disease. Liquid-based cytology (LBC) samples were used to optimize a dual ProExC/Ki-67 immunofluorescence staining protocol for SurePath-fixed cells. The simultaneous and automated detection of these biomarkers was performed using the BD Pathway 435 system. The ability of high-content imaging to detect dysplastic cervical cells was assessed using keratinocytes spiked with immunopositive SiHa cells and a high-grade squamous intraepithelial lesion (HSIL) LBC sample. The percentages of Ki-67- and ProExC-immunopositive objects correlated significantly with the percentages of spiked SiHa cells. The dysplastic cells of the HSIL sample could be detected using high-content cell analysis. In conclusion, high-content imaging allows the simultaneous and automated detection of Ki-67- and ProExC-immunopositive dysplastic cells in LBC specimens.