Digital microscopy produces high resolution digital images of pathology slides. Because no acceptable and effective control of color reproduction exists in this domain, there is significant variability in color reproduction of whole slide images. Guidance from international bodies and regulators highlights the need for color standardization. To address this issue, we systematically measured and analyzed the spectra of histopathological stains. This information was used to design a unique color calibration slide utilizing real stains and a tissue-like substrate, which can be stained to produce the same spectral response as tissue. By closely mimicking the colors in stained tissue, our target can provide more accurate color representation than film-based targets, whilst avoiding the known limitations of using actual tissue. The application of the color calibration slide in the clinical setting was assessed by conducting a pilot user-evaluation experiment with promising results. With the imminent integration of digital pathology into the routine work of the diagnostic pathologist, it is hoped that this color calibration slide will help provide a universal color standard for digital microscopy thereby ensuring better and safer healthcare delivery.