Introduction and rationale Myocardial infarction (MI) can be surgically induced in mice. Accurate measurement of infarction size (IS) is regularly used as an experimental readout. Established methods for IS consist of macroscopic or histological staining of the left ventricle (LV). These methods do not allow for further molecular analysis and do not characterize the functional state of the LV. Echocardiographic analysis of the LV is often performed in MI studies, yet most studies rely on 2D-B-Mode or M-Mode imaging. Here, the extent of the infarction can easily be misjudged. Therefore, we wanted to develop a new strategy to reliably estimate IS in echocardiography. Methods and results Echocardiography was performed at baseline conditions and 7 days after MI induction using a Vevo 3100 sonography device with a MX550S transducer (slice thickness for 4D datasets: 150μm). For 4D strain analysis, a custom-made software tool was used to trace the endocardial surface of the LV. The method was checked for inter-observer reliability which was excellent for volumetry and estimation of systolic function (correlation of ejection fraction between observers: r=0.9414, p<0.0001). Surface area strain was computed and the area that displayed a surface area strain >−10% was considered infarcted. IS measured by 4D surface area strain was correlated with ejection fraction (r=−0.510, p=0.002) and IS estimated by triphenyl tetrazolium chloride staining (r=0.582, p=0.005). Conclusion 4D strain analysis based estimation of IS is a reliable method for functional and morphological characterization of infarcted hearts. Other than established IS measurements, it allows for additional molecular analyses within the same individuum and can be used to monitor growth or reduction of the akinetic area over time. It therefore emerges as a powerful tool for murine MI studies. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): DFG - GRK 2407 (360043781)