Soft lithography with microfabricated molds is a widely used manufacturing method. Recent advancements in 3D printing technologies have enabled microscale feature resolution, providing a promising alternative for mold fabrication. It is well established that the curing of PDMS is influenced by parameters such as temperature, time, and curing agent ratio. This study was conducted to address inconsistencies in PDMS curing observed when using different 3D-printed mold materials during the development of a Lab-on-a-Chip (LoC) system, which is typically employed for investigating the effect of hydrodynamic cavitation on blood clot disintegration. To evaluate the impact of mold material on PDMS curing behavior, PDMS was cast into molds made from polylactic acid (PLA), polyethylene terephthalate (PET), resin, and aluminum, and cured at controlled temperatures (55, 65, and 75 °C) for various durations (2, 6, and 12 h). Curing performance was assessed using Soxhlet extraction, Young’s modulus calculations derived from Atomic Force Microscopy (AFM), and complementary characterization methods. The results indicate that the mold material significantly affects PDMS curing kinetics due to differences in thermal conductivity and surface interactions. Notably, at 65 °C, PDMS cured in aluminum molds had a higher Young’s modulus (~1.84 MPa) compared to PLA (~1.23 MPa) and PET (~1.17 MPa), demonstrating that the mold material can be leveraged to tailor the mechanical properties. These effects were especially pronounced at lower curing temperatures, where PLA and PET molds offered better control over PDMS elasticity, making them suitable for applications requiring flexible LoC devices. Based on these findings, 3D-printed PLA molds show strong potential for PDMS-based microdevice fabrication.