Background: In vitro endothelial cell culture is widely used to study angiogenesis. Histomicrographic images of cell networks are often analyzed manually, a process that is time-consuming and subjective. Automated tools like ImageJ (NIH) can assist, but are often slow and inaccurate. Additionally, as endothelial networks grow more complex, traditional architectural metrics may not fully reflect network maturity. To address these limitations, we developed tubuleTracker, a software tool that quantifies endothelial network architecture and maturity rapidly and objectively. Methods: Human umbilical vein endothelial cells were cultured in an extracellular matrix, and 54 images were acquired using phase contrast microscopy. Each image was analyzed manually by three independent reviewers, and by both ImageJ and tubuleTracker. Key metrics included tubule count, total length, node count, tubule area, and vessel circularity. In parallel, trained scientists rated each image for angiogenesis maturity on a 1-5 scale (1 = most mature). Results: Analysis time per image differed significantly: manual (8 min), ImageJ (58+/-4 s), and tubuleTracker (6+/-2 s) (p<0.0001). Significant differences were also found in tubule count (manual 168+/-SD, tubuleTracker 92+/-SD, ImageJ 433+/-SD), length, and node count (all p<0.0001). tubuleTracker's metrics varied significantly across angiogenesis maturity scores, including tubule count, length, node count, area, and circularity (all p<0.0001). Conclusions: tubuleTracker was faster and more consistent than both manual and ImageJ-based analysis. Vessel circularity proved especially effective in capturing angiogenesis maturity. tubuleTracker is available as free shareware for the biomedical research community.