Cuticle scales are the most obvious feature of a hair fibre's outer surface and much research has focused on assessing the influence of surface topography on the associated hair fibre's properties. However, much of the research has either been qualitative or, if quantitative, employed relatively laborious analytical techniques to establish the necessary statistical robustness. In this study, we report on the application of a 3D image analysis package capable of producing 3D data from multiple 2D scanning electron microscope (SEM) images of hair fibres. Analysis of the surface profile can be carried out quickly and accurately, enabling quantification of the scale structure. To validate the novel technique and ensure that the scale heights measured were indeed accurate and reproducible, extensive calibration of the SEM and the 3d software has been performed. In addition, scale heights on a single hair have been determined by using atomic force microscopy (AFM) and the results compared with analogous data produced from the same scale edges using the 3D image analysis technique. The data obtained indicate that a relatively quick, accurate and viable method to determine scale height in keratin fibres has been established. A further 3D-SEM analysis method has been developed which allows individual scales to be monitored and for the cuticular scale height to be quantified after repeated 'smoothing' treatments.