Optical slice microscopy is commonly used to characterize the morphometric features of 3D cellular cultures, such as in vitro vascularization. However, the quantitative analysis of those structures is often performed on a single 2D maximum intensity projection image, limiting the accuracy of data obtained from 3D cultures. Here, we present a protocol for the quantitative analysis of z stack images, utilizing Fiji, Amira, and WinFiber3D. This protocol facilitates the in-depth examination of vascular-like structures within 3D cell culture models. For complete details on the use and execution of this protocol, please refer to Koch et al. (2020).