Blood flow patterns and endothelium interactions are of great importance for the progression of cardiovascular diseases, thus quantitative analysis of blood flow dynamics at the microcirculation level of great relevance. Regulatory mechanisms mediated by blow flow have been studied in detail using in vitro approaches. However, these mechanisms have not been completely validated in vivo due to technical limitations involved in the quantification of flow patterns in the mcirocirculation with the required level of detail. Intravital microscopy combined with high‐speed video recordings has been used for the analysis of blood flow in small blood vessels of chronic and acute experimental animal tissue preparations. This tool is used to study the interaction between the flowing blood and the vessel walls of arterioles and venules with great temporal and spatial resolution. Our objective is to develop a simple and robust cross‐correlation algorithm for the automatic analysis of high‐speed video recordings of microcirculatory blood flow. The algorithm has been validated using in vitro (glass microchannels) and in vivo (window chamber models) systems. Results indicate that the algorithm’s ability to estimate the velocity of local red blood cells (RBCs) as a function of blood vessel radius and time is highly accurate.The algorithm could be used to explore dynamic changes in blood flow under different experimental conditions including a wide range of flow rates and hematocrit levels. The algorithm can also be used to measure volumetric flow rates, radial velocity profiles, wall shear rate (WSR), and wall shear stress (WSS). Several applications are presently explored, including the analysis of velocity profiles in the branches of arterial bifurcations. Our work demonstrates the robustness of the image cross‐correlation technique in various flow conditions and elucidates its potential application for in vivo determination of blood flow dynamics in the microcirculation. Grant Funding Source : Supported by: NIH R01 HL52684, R01 HL064395 and R01 HL062318 and ARMY: W81XWH‐11‐2‐0012