Color calibration of digital microscope images of stained biological slides may use a special slide (called a calibration slide) with known color samples that are usually filters. To evaluate quantitatively how well the calibration has been done by a particular system, another set of different known samples is needed, with transmittance spectra that are typical of stained biological material. A slide with such colors comprises what is called a reference slide. In this article, a method is described for selecting the colors for such a slide. A reference slide was created by this prescription and then used to evaluate two embodiments of a color management system for several microscope and illumination combinations. It was found that a set of 20 reference colors spans the gamut of colors produced by a particular kind of stain. Using the reference slide, Datacolor's ChromaCal system performance was quantitatively evaluated. The performance is robust to change of microscope and illumination and does not degrade much when a tungsten light is increased in intensity (hence in correlated color temperature) with only one initial calibration at a single color temperature followed by automatic white balancing of a traditional sort. (c) 2014 Wiley Periodicals, Inc. Col Res Appl, 40, 577-584, 2015