A discussion is provided of rapid prototyping and characterization of microfluidic devices with laser machining and advanced confocal microscopy. Topics covered are structural characterization, flow characterization, prototyping, and biochemical sensor. Microfluidic development has much promise and is still nascent. Work required of scientists and engineers includes understanding interactions between structure and flow at the micrometer scale and design of devices to reach the performance desired. Both sets of tasks require the ability to characterize structure and flow and also to fabricate structures that range widely in shape and size. Advanced confocal microscopy provides fast, precise structural characterization and is compatible with a range of flow characterization techniques. Laser ablation machining is an excellent choice for iterative, one-of-a-kind requirements of prototype fabrication in a microfluidic development environment. Advanced confocal microscopy can characterize structure with submicrometer resolution. When used with other techniques, it also visualizes flow at the micrometer scale, and laser ablation offers the ability to fabricate microscale structures of almost any shape. Confocal microscopy adds a third, vertical dimension to the two-dimensional imaging obtained from conventional microscopy, and the purpose of microfluidic structure is control of microfluidic flow. The structural and flow characterization techniques must be mutually compatible and implemented on a common platform. Both must be able to observe functioning devices without alteration between structure and flow characterizations. For microfluidics, this generally requires an ability to observe flow in closed subsurface structures.