We report the design, fabrication, and characterization of practical microfluidic valves fabricated using laser printer lithography. These optofluidic valves are opened by directing optical energy from a solid-state laser, with similar power characteristics to those used in CD/DVD drives, to a spot of printed toner where localized heating melts an orifice in the polymer layer in as little as 500 ms, connecting previously isolated fluidic components or compartments. Valve functionality, response time, and laser input energy dependence of orifice size are reported for cyclo-olefin polymer (COP) and polyethylene terephthalate (PET) films. Implementation of these optofluidic valves is demonstrated on pressure-driven and centrifugal microfluidic platforms. In addition, these “one-shot” valves comprise a continuous polymer film that hermetically isolates on-chip fluid volumes within fluidic devices using low-vapor-permeability materials; we confirmed this for a period of one month. The fabrication and integration of optofluidic valves are compatible with a range of polymer microfabrication technologies and should facilitate the development of fully integrated, reconfigurable, and automated lab-on-a-chip systems, particularly when reagents must be stored on chip for extended periods, e.g. for medical diagnostic devices, lab-on-a-chip synthetic systems, or hazardous biochemical analysis platforms.