MOTIVATION: Cardiomyocytes derived from stem cells are closely followed, notably since the discovery in 2007 of human induced pluripotent stem cells (hiPSC). Cardiomyocytes (hiPSC-CM) derived from hiPSC are indeed more and more used to study specific cardiac diseases as well as for developing novel applications such as drug safety experiments. Robust dedicated tools to characterize hiPSC-CM are now required. The hiPSC-CM morphology constitutes an important parameter since these cells do not demonstrate the expected rod shape, characteristic of native human cardiomyocytes. Similarly, the presence, the density and the organization of contractile structures would be a valuable parameter to study. Precise measurements of such characteristics would be useful in many situations: for describing pathological conditions, for pharmacological screens or even for studies focused on the hiPSC-CM maturation process. RESULTS: For this purpose, we developed a MATLAB based image analysis toolbox, which gives accurate values for cellular morphology parameters as well as for the contractile cell organization. AVAILABILITY AND IMPLEMENTATION: To demonstrate the power of this automated image analysis, we used a commercial maturation medium intended to promote the maturation status of hiPSC-CM, and compare the parameters with the ones obtained with standard culture medium, and with freshly dissociated mouse cardiomyocytes. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.