Objective To establish a rapid and efficient method for determining right ventricular pressure in mice without the aid of a ventilator and explore the measurement techniques. Methods Twenty-three C57BL/ 6 J mice (8 weeks old) were randomly divided into two groups: control group ( n = 12) and hypoxic group ( n = 11). The hypoxic group was reared in a hypoxic chamber (10% O2 _{) for 4 weeks to prepare the pulmonary hypertension model, while the control group was reared in normal air; otherwise, the conditions were the same. Then, we connected a multi-channel physiological instrument and measured right ventricular pressure in mice with a 0. 6×25 mm disposable infusion needle by puncturing the intercostal muscle directly when the chest cavity was closed. Thoracotomy was performed to verify the puncture point. Next, the heart was separated, the right ventricle (RV), and left ventricular and septum (LV + S) were weighed, and the right ventricular hypertrophy index ( RVHI) was calculated. Pathological sections of left lung were taken and stained with hematoxylin-eosin and α-smooth muscle actin to observe the structural changes of pulmonary artery. Results A right ventricular pressure curve was obtained by directly puncturing the right ventricle through the intercostal muscle, and the whole process was completed within 3- 4 minutes; the success rate of RVSP measurement was 95. 6% ( 1 failed in the control group). The average RVSP and RV/ (LV + S) of the hypoxia group were 30. 48±2. 95 mmHg and 35. 31±3.78%, which were higher than those of the control group with 17. 50±3. 40 mmHg and 23. 14±4. 29% ( both P < 0. 05), respectively. In the hypoxic group, the pulmonary arterioles were thickened and the smooth muscle of the tunica media proliferated. Conclusions The method of measuring the right ventricular pressure curve of mice by intercostal muscle puncture and keeping the animal chest closed without ventilator-assisted ventilation is simple and fast; it can rapidly assess the hemodynamic state of the right ventricle of mice with a high success rate and is worthy of being popularized.}