Much research is being directed toward investigating links between quantitative characteristics of the retinal vasculature and a variety of outcomes to identify biomarkers. The interest for retinal biomarkers lies in the fact that the retina is easily observed via fundus photography. Outcomes considered for research of biomarkers in the literature include conditions such as diabetes and lacunar stroke, and also cognitive performance and genetic expression [1][2][3][4][5]. The need for measuring large volumes of images, needed to power biomarker discovery studies, makes semiautomatic software systems desirable. This chapter reports recent algorithms developed by the VAMPIRE group for vasculature detection and quantification, including recent developments on landmark detection. We focus on accuracy and validation issues, and, importantly, the conditions for comparing meaningful results from different algorithms. This work is a part of VAMPIRE (Vasculature Assessment and Measurement Platform for Images of the REtina), which is an international collaboration growing a software suite for automatic morphometric measurements of the retinal vasculature.