The performance of Cs+, C60+ and Arn+ (with n ≈ 1700) sputtering ions have been compared for depth profiling multilayer films made from three evaporated phenylalanine layers sandwiched between four thicker evaporated tyrosine layers. Using Cs+, the ion signals and depth resolution degrade with depth and were significantly affected beyond a 200-nm depth. The depth profiling quality was more successful using C60+. However, in this case, the depth resolution and the layer width values still degrade with the sputtered depth and are particularly poor after reaching a depth of about 400 nm. When Ar1700+ clusters were used, a depth resolution as low as 6 nm was obtained, and this value never exceeds 9 nm. Moreover, the experimental layer width is found to be of the same order of magnitude as the real value. Copyright © 2012 John Wiley & Sons, Ltd.