Image analysis methods can be used to quantify intercapillary distance and further histological measures of microcirculation-associated marker distribution. Compared with manual methods, greater numbers of measurements can be performed in less time and observer-induced bias may be reduced. Even so, the equipment required for image analysis is often expensive and procedures established on one system may not necessarily be transferable to others. Standardization of image analysis for a series of microcirculation-associated parameters can be greatly enhanced by high-performance freeware image analysis software such as ImageJ. In the present study, a simple and reproducible method for both the calculation of intercapillary distances and microcirculation-associated marker expression that can be performed entirely in ImageJ is demonstrated. Possible applications include the study of endogenous markers associated with tumor hypoxia (e.g., CA IX) or the assessment of proliferation/apoptosis in relation to the vasculature in immunohistochemical double stains.