Objective To assess residual protein on dental instruments cleaned in general dental practice by manual, manual plus ultrasonic and automated washer disinfector (AWD) processes.Design and setting Instruments submitted by 30 dental surgeries in the South West of England.Subjects (materials) and methods Instruments analysed were matrix bands, associated retaining clips, diamond and stainless steel burs, extraction forceps and hand scalers. Each instrument was visually assessed under magnification for residual debris. Residual protein was extracted by immersion in detergent and sonication. A collection of used but uncleaned instruments of each type (n = 177) was also analysed for adherent protein using ophthalaldehyde/N-acetylcysteine reagent.Main outcome measures Residual protein levels allowed comparisons to be made on the effectiveness of different cleaning processes.Results One thousand, three hundred and four instruments were analysed. Observational data demonstrated several shortcomings in cleaning chemistries and operation of the AWD. For uncleaned instruments, median residual protein levels ranged from 0.4 μg (stainless steel burs) to 462 μg (extraction forceps). Following manual washing, median protein levels ranged from 0.3-78 μg; for manual plus ultrasonic washing, levels ranged from 9-39 μg and AWD levels ranged from 0.3-27 μg. Manual washing combined with ultrasonic cleaning was significantly less effective than the other two processes (p <0.008). AWDs reduced the variability in the cleaning process. No correlation was found between visual scoring and residual protein determination.Conclusion(s) There was a wide variation in residual protein levels both within and between different methods and instruments and this underlines the complexity of this process.