Cardiovascular disease (CVD) is the number one cause of death worldwide. This condition resulted in huge research on CVD increasing the need for animal models suitable for in vivo research. Daphnia and zebrafish are good animal models for cardiovascular research due to their relative body transparency and easy culture property. Several methods have been developed to conduct cardiac performance measurement in Daphnia and zebrafish. However, most of the methods are only able to obtain heartbeat rate. The other important cardiac endpoints like stroke volume, ejection fraction, fraction shortening, cardiac output, and heartbeat regularity must use other programs for measurement. To overcome this limitation, in this study, we successfully developed a one-stop ImageJ-based method using kymograph macros language that is able to obtain multiple cardiac performance endpoints simultaneously for the first time. To validate its utility, we incubated Daphnia magna at different ambient temperatures and exposed zebrafish with astemizole to detect the corresponding cardiac performance alterations. In summary, the kymograph method reported in this study provides a new, easy to use, and inexpensive one-stop method obtaining multiple cardiac performance endpoints with high accuracy and convenience.