During the last decade, oral cytology has once again become the focus of scientific research. This new interest is due to the introduction of a cytobrush for cell collection as well as a computer-assisted analysis (Oral CDx®). Although promising, the sensitivity and specificity of conventional oral brush cytology remains limited. To circumvent the problems and improve the accuracy, various adjunctive analytical methods have been attempted. DNA analysis, immunocytochemical and molecular analysis are suggested methodological cytology approaches to improve the validity of oral brush cytology. An increase in sensitivity (up to 100%) and specificity (up to 100%) of oral brush biopsy has been reported on localized pre-malignant and malignant lesions. Oral brush biopsy probably will not replace histopathology in the definitive diagnosis of oral cancer, but it might be valuable for the prevention of misdiagnosis of clinically doubtful oral lesions and for the monitoring of lesions that might proceed on to oral cancer.