AbstractImage cytometry is the process of converting image data to flow cytometry-style plots, and it usually requires computer-aided surface creation to extract out statistics for cells or structures. One way of dealing with structures stained with multiple markers in three-dimensional images, is carrying out multiple rounds of channel co-localization and image masking before surface creation, which is cumbersome and laborious. We propose the application of the hue-saturation-brightness color space to streamline this process, which produces complete surfaces, and allows the user to have a global view of the data before flexibly defining cell subsets. Spectral compensation can also be performed after surface creation to accurately resolve different signals. We demonstrate the utility of this workflow in static and dynamic imaging datasets of a needlestick injury on the mouse ear, and we believe this scalable and intuitive approach will improve the ease of performing histocytometry on biological samples.