Systems and methods for analyzing vessels in multiplexed images include detecting large vessels using a spoke feature detection method, detecting long and narrow vessels using a line feature detection method, detecting smaller vessels using rolling-ball filtering and binary image operations to generate a mask, and evaluating any contour polygons resulting from these operations using quality measurements and other thresholds. Maturity determination and nuclei detection are also performed, resulting in an output of vessel characteristics and co-locations enabling enhanced analysis of multispectral images.