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Abstract Background To understand the relationship between myocardial contractility and external stimuli, detecting ex vivo myocardial contractility is necessary. Methods We elaborated a method for contractility detection of isolated C57 mouse papillary muscle using Myostation‐Intact system under different frequencies, voltages, and calcium concentrations. Results The results indicated that the basal contractility of the papillary muscle was 0.27 ± 0.03 mN at 10 V, 500‐ms pulse duration, and 1 Hz. From 0.1 to 1.0 Hz, contractility decreased with an increase in frequency (0.45 ± 0.11–0.10 ± 0.02 mN). The voltage‐initiated muscle contractility varied from 3 to 6 V, and the contractility gradually increased as the voltage increased from 6 to 10 V (0.14 ± 0.02–0.28 ± 0.03 mN). Moreover, the muscle contractility increased when the calcium concentration was increased from 1.5 to 3 mM (0.45 ± 0.17–1.11 ± 0.05 mN); however, the contractility stopped increasing even when the concentration was increased to 7.5 mM (1.02 ± 0.23 mN). Conclusions Our method guaranteed the survivability of papillary muscle ex vivo and provided instructions for Myostation‐Intact users for isolated muscle contractility investigations.