Objective: Color perception is an important variable in detecting and assessing oral conditions. The aim was to investigate clinicians' perception of toluidine blue (Tblue) staining compared to digital color analysis, which may impact mucosal lesion detection, affect the decision to biopsy, and biopsy site selection. Method and Materials: Four oral lesions were stained with Tblue. Digital color analyses of eight areas on each image were completed and were considered as "gold standard" (GS). Twenty specialists ranked these areas according to their perceived intensity of blue stain in two sessions. Results: Consistency between GS and observers rankings was 0.8791. However, more than half of the observers inaccurately perceived the intermediate blue tones. Overall interobserver agreement was 0.8714; stability between two sessions decreased to 45% for intermediate tones. Conclusion: Assessing the equivocal blueness of an oral mucosal lesion in clinical settings may vary due to variation in visual perception. A digital method for objective color analysis in clinical practice may be used to eliminate this deficiency by implementing a mathematical formula. Keywords: color perception, oral cancer, oral diagnosis, oral medicine, toluidine blue