Microfluidic paper-based analytical devices (mPADs) have gained significant attention in recent years for applications ranging from clinical diagnostics to environmental testing. However, separation on mPADs remains challenging to implement, particularly in complex samples. This has revived interest in revisiting paper chromatography and paper electrophoresis in mPADs to address these needs. Here, laminated Parafilm-paper (l-paper) is applied to fabricate electrophoretic devices. This approach yields a free-standing channel, leading to improved peak resolution relative to previous electrophoretic separations in traditional wax-printed mPADs. Major factors influencing the separation, including Joule heating, electroosmotic flow, and electrophoretic mobility, were investigated. As a result of paper’s high ratio of surface area (78%) to pore volume (22%) resulting in slow heat dissipation, a usable applied field strength range of 0–200 V cm−1 was employed to avoid Joule heating. The electroosmotic flow of the system was found to be 2.5 × 10−5 ± 7.7 × 10−7 cm2 V−1 s−1 and the electrophoretic mobility of chlorophenol red was 1.2 × 10−4 ± 7.7 × 10−7 cm2 V−1 s−1. Basic separation protocols were optimized using colorimetric detection of chlorophenol red and indigo carmine dyes as representative molecules. Paper type, channel width, and applied potential were then used to optimize the separations. Addition of an injection port to the device improved resolution and reduced peak broadening. Finally, the separation of fluorescein isothiocyanate (FITC) and l-glutamic acid (Glu) labeled with FITC, was successfully carried out using the l-paper electrophoretic device. Imaging with a microscope was found to achieve reduced peak broadening and increased resolution relative to imaging with a mobile camera, due to elimination of background signal, achieving a 72 ± 4% conjugation of Glu and FITC.