Techniques for microfluidic channel fabrication in soda-lime glass and fused quartz using femtosecond laser ablation and ablation in conjunction with polymer coating for surface roughness improvement were tested. Systematic experiments were done to characterize how process variables (laser fluence, scanning speed and focus spot overlap, and material properties) affect the machining feature size and quality. Laser fluence and focus spot overlap showed the strongest influence on channel depth and roughness. At high fluence, the surface roughness was measured to be between 395 nm and 731 nm RMS. At low fluence, roughness decreased to 100 nm–350 nm RMS and showed a greater dependence on overlap. The surface roughness of laser ablation was also dependent on the material properties. For the same laser ablation parameters, soda-lime glass surfaces were smoother than fused quartz. For some applications, especially those using quartz, smoother channels are desired. A hydroxyethyl methacrylate (HEMA) polymer coating was applied and the roughness of the coated channels was improved to 10–50 nm RMS.