Evaluation of cardiomyocyte contraction using hiPSC-CMs holds great promise to predict drug-induced heart failures in vitro. However, it has been difficult to detect the positive inotropic effects of drug using hiPSC-CMs. Due to the immature property, hiPSC-CMs do not show alignment. In this study, we evaluated whether hiPSC-CM with alignment can detect the drug-induced positive inotropic effects. We used hiPSC-CM (iCell Cardiomyocyte, FCDI), which were cultured using commercially available plates for alignment. After long-term culture, we found that the aligned culture improved the basal contraction velocity, which were measured by motion vector analysis (SI8000, Sony). To understand the molecular mechanisms of the contraction improvement, we performed next-gene sequencing analysis. Gene expression levels of ion channels and metabolism-related factors were up-regulated by alignment. In addition, we observed that isoproterenol enhanced contraction velocity in hiPSC-CMs with alignment in a dose-dependent manner. These results suggests that the aligned culture of hiPSC-CMs facilitates the maturation and the responses to isoproterenol.