Utilizing micro-electro-mechanical-systems (MEMS) techniques and a solvent-assisted bonding process, a new generation of diffuser peristaltic polymethylmethacrylate (PMMA) micropumps was optimized and fabricated. The main purpose of this study is to compare the performance of optimized and un-optimized micopump which have the same diffuser throat/inlet area (i.e. 16000? μ2). Furthermore, an additional optimized design which has smaller diffuser inlet area was considered to validate and analyze the effect of diffuser inlet area to the micropump performance. The experimental results were validated by comparing with previous generation which had not been optimized the diffuser element. Specifically, the experimental results showed that, with similar diffuser element inlet area (i.e. 160000 μm2), with and without optimized micropumps yield maximum flow rates of 246.4 μL/min and 194.8 μL/min, respectively. Furthermore, it is shown that the back pressure in the optimized micropump is 6.9 kPa, while that in the un-optimized pump is 5.69 kPa. The effect of diffuser element throat/inlet area to pump flow rate and back pressure was investigated by comparing the experimental results of two optimized designs, one with 80 μm × 80 μm and the other with 127 μm × 127 μm cross-sectional area. The results indicated that, the design with larger inlet area gave higher flow rate. However, the rate of reduction in the maximum flow rate with increasing back-pressure increases at the higher inlet area design, which is due to the greater pressure dissipation/loss associated with a larger channel cross-sectional area.