In basic and translational cardiovascular research, the in vivo assessment of heart function represents a critical step towards a better definition of an animal model, a molecular target, or a potential pharmacological approach. While echocardiography is the most used imaging method as it is readily available and relatively cheap, multi-modal approaches have been developed to merge anatomical and functional parameters with molecular imaging data. From the gold standard cardiac MRI to the novel ultrasound/photoacoustic hybrid platforms, cardiovascular scientists can now use tons of imaging techniques to better dissect molecular and pathophysiological aspects of their interest, often at once. Even though cardiovascular imaging is widely used in many experimental cardiology labs, no standardized protocols for images acquisition and analysis exist. A standardization of heart and vascular assessment using current imaging approaches is essential to provide reproducible and reliable in vivo data and to improve the overall research quality. This Methods Collection aims to gather echocardiographic, MRI, and multi-modal imaging protocols to serve as a compendium for cardiovascular scientists that are using or will use imaging in the future. Main areas of interest may include (but are not limited to): functional and anatomical assessment of the four chambers in small and large animal models, study of diastole, strain analysis, 3D & 4D methods, molecular imaging, and vascular imaging.