Microvascular networks can be modelled as a network of connected cylinders. Presently, however, there are limited approaches with which to recover these networks from biomedical images. We have therefore developed and implemented computer algorithms to geometrically reconstruct three-dimensional (3D) retinal microvascular networks from micrometre-scale imagery, resulting in a concise representation of two endpoints and radius for each cylinder detected within a delimited text file. This format is suitable for a variety of purposes, including efficient simulations of molecular delivery. Here, we detail a semi-automated pipeline consisting of the detection of retinal microvascular volumes within 3D imaging datasets, the enhancement and analysis of these volumes for reconstruction, and the geometric construction algorithm itself, which converts voxel data into representative 3D cylindrical objects.