We present a flexible microfluidic channel fabrication platform that can be used to develop microfluidic chips. A DPSS (diode pumped solid state) frequency quadrupled (λ = 266 nm, the UV system) Nd:YAG laser and a CO2 laser (λ = 10.6 μm, the IR system) are compared for their ablation capability on quartz and glass. We have also compared their performance in developing microfluidic chips. The resultant surface quality, including microcracking, debris, and distortion, is examined by SEM and a surface profiler. In these systems, users design microfluidic patterns by commercial software. The pattern is then transferred to a CNC stage for trenching. The microfabrication process can be completed in several minutes. Without the need to fabricate photomask for patterning, the development time can be reduced from weeks to hours. In addition, the substrate size is not limited by the dimension of the photomask. Asymmetric trenches demonstrating the machining capability of these systems have been fabricated by these systems. The minimal feature for the IR system and the UV system is 140 μm and 5 μm, respectively. These systems are very powerful for rapid glass microfluidic chip development.