This study presents a double-side diaphragm peristaltic pump for efficient medium transport without the unwanted backflow and the lagging effect of a diaphragm. A theoretical model was derived to predict the important parameter of the micropump, i.e., the motion of the valves at large deformations, for a variety of air pressures. Accordingly, we proposed an easy and robust design to fabricate a Polydimethylsiloxane (PDMS)-based micropump. The theoretical model agrees with a numerical model and experimental data for the deformations of the PDMS membrane. Furthermore, variations of the generated flow rate, including pneumatic frequencies, actuated air pressures, and operation modes were evaluated experimentally for the proposed micropumps. In future, the theoretical equation could provide the optimal parameters for the scientists working on the fabrication of the diaphragm peristaltic pump for applications of cell-culture.