ABSTRACTClassically, the limit for optical machining is on the order of the wavelength of the incident light. However, by taking advantage of precise, nonlinear damage mechanisms that occur for femtosecond laser pulses, damage can be achieved on a scale an order of magnitude lower, allowing precise removal of very small amounts of material to produce holes mere tens of nanometers wide. Femtosecond laser nanomachining can be carried out in a variety of dielectrics, and in transparent substrates machining can be sub-surface, in contrast to other nanomachining techniques such as using an electron beam or focused ion beam. We focus on the use of glass, as it is in many ways an ideal material for use in biological applications due to its chemical, optical, electrical and mechanical properties. By precisely placing laser pulses in glass, three dimensional nano and microfluidic channels and devices can be formed including nozzles, mixers, and separation columns. Recent advances in this technique allow the formation of high aspect ratio nanochannels from single pulses, thus helping address the fabrication speed limitations presented by serial processing. These nanochannels have a range of applications including the fabrication of nanoscale pores and nanowells that may serve as vias between fluidic channels, or from channels to a surface. These nanochannels have applications as a standalone technique for fabrication of nanopores and nanowells, but can also complement other fabrication techniques by allowing precisely placed jumpers that can connect channels that are out of plane. We discuss applications for diagnostic microfluidic devices, and basic cell biology research.