This chapter describes the methods and specific procedures used to fabricate microstructures by soft lithography. These techniques are useful for the prototyping of devices useful for applications in biotechnology. Fabrication by soft lithography does not require specialized or expensive equipment; the materials and facilities necessary are found commonly in biological and chemical laboratories in both academia and industry. The combination of the fact that the materials are low-cost and that the time from design to prototype device can be short (< 24 h) makes it possible to use and to screen rapidly devices that also can be disposable. Here we describe the procedures for fabricating microstructures with lateral dimensions as small as 1 μm. These types of microstructures are useful for microfluidic devices, cell-based assays, and bioengineered surfaces.