Quantification of the tricuspid valve (TV) leaflets mechanical strain is important in order to understand valve pathophysiology and to develop effective treatment strategies. Many of the traditional methods used to dynamically open and close the cardiac valves in vitro via flow simulators require valve dissection. Recent studies, however, have shown that restriction of the atrioventricular valve annuli could significantly change their in vivo deformation. For the first time, the porcine valve leaflets deformation was measured in a passive ex vivo beating heart without isolating and remounting the valve annuli. In particular, the right ventricular apexes of porcine hearts (n = 8) were connected to a pulse-duplicator pump that maintained a pulsatile flow from and to a reservoir connected to the right atrium and the pulmonary arteries. This pump provided a right ventricular pressure (RVP) waveform that closely matched physiological values, leading to opening and closure of the tricuspid and pulmonary valves (PVs). At the midsection of the valve leaflets, the peak areal strain was 9.8 ± 2.0% (mean±standard error). The peak strain was 5.6 ± 1.1% and 4.3 ± 1.0% in the circumferential and radial directions, respectively. Although the right ventricle was beating passively, the leaflet peak areal strains closely matched the values measured in other atrioventricular valves (i.e., the mitral valve (MV)) in vivo. This technique can be used to measure leaflet strains with and without the presence of valve lesions to help develop/evaluate treatment strategies to restore normal valve deformation.