Tooth bleaching has become an increasingly popular method to lighten tooth colour and improve tooth aesthetics. Accurate colour determination prior and after the procedure is pivotal to the treatment. Objectives: This in vitro study evaluates three methods of tooth colour determination: the eye and VITAPAN 3D-MASTER shade guide (EYE) and two electronic instruments the Revised Shade Guide (RSG) and VITA Easyshade machine (VITA). Three tooth types usually associated with bleaching: incisors, canines and premolars were assessed. Methods: Extracted human teeth which had been stored in 1% thymol at 8°C were used for this study. One hundred and twenty nine teeth of a colour range typically required for bleaching were selected, scaled and polished. There were 45 premolars, 18 canines and 36 incisors. Each tooth was coded, randomly grouped into replicates of 11 teeth and measured for colour while damp. EYE measurements were done using the same lighting conditions each time. VITA and RSG instruments were self calibrated prior to each reading session as required. Intra examiner variability was tested using 30 additional teeth, which were read, 4 times at intervals during the assessment. Data was recorded on a spread sheet and tested using the Friedman two way randomized block design with P<0.05. Results: Intra-examiner variability was at P=0.18. Statistical testing showed no significant difference between the three colour determination methods for all three tooth types. However, both electronic colour determination instruments were cumbersome, difficult to use, capricious and time consuming. The colour readings bounced between a wide variety of colours for an individual tooth and at times would give no readings at all. Conclusion: The eye is more efficient and as good a colour discriminator as the two expensive colour determination instruments tested.