Leaf vein density (LVD) is a critical trait linked to leaf hydraulic efficiency, commonly quantified using an automated tool—LEAF GUI, which analyzes vein structures via pixel-based algorithms. However, concerns persist about its accuracy for species with complex hierarchical venation networks. To evaluate the reliability of LEAF GUI, we compared its LVD measurements against those from ImageJ—a manual tracing platform renowned for its precision—using leaf specimens from nine Magnoliaceae species. Paired t-tests revealed no statistically significant differences between the two methods (P = 0.534), demonstrating comparable accuracy under standardized conditions. However, LEAF GUI’s reliability is constrained by its reliance on laborious threshold calibration and stringent image quality standards. For studies prioritizing precision, especially in taxa with heterogeneous or low-resolution samples, we recommend ImageJ as the standard approach. Its manual tracing protocol achieves consistency in resolving complex vein networks, balancing analytical rigor with adaptability to diverse sample conditions