Recent multicolor multiphoton microscopy schemes are opening the way to high quality fluorescence color imaging at near-micrometric resolution over virtually unlimited volumes of brain tissue. However, unavoidable artifacts related to depth, chromatic aberration or anisotropic resolution affect the resulting images and can bias measurements and quantifications. Here, we prove the possibility of performing precise automated quantitative measurements on such tridimensional multicolor images, taking as a model fluorescently labeled axons belonging to an auditory tract in the mouse brainstem. We present an analysis pipeline to compute the diameter of these axons based on the calculation of distance in HSV color space, its binarization using a random walker algorithm and skeleton extraction by distance transform. We measure a biologically meaningful difference of about 500 nm before and after the axons enter their target nucleus where they form synaptic connections, proving the robustness of our pipeline with respect to the aforementioned limitations. This demonstrates the ability of sub-micrometric measurements across tissue depths of 0.5 mm with multicolor multiphoton microscopy.