Hydrostatic pressures of 102 to 103 atm affect a range of cellular processes, including motility, cell division, nutrient uptake, fermentation, translation and transcription, protein synthesis, and ultimately viability. In order to perform the real time monitoring of pressure effects, we present a micro-perfusion system designed for spectroscopic measurements on cellular systems under high pressure. The system consists of an optically-compatible pressure chamber and interchangeable fluid reservoirs. Perfusion is achieved using a dual pressure-generator configuration, where one positive-displacement generator is compressed while the other is retracted, thus maintaining a pressurized volume while achieving fluid flow. Control over perfusion rates (typically in the 10 μl/s range) and the ability to change between fluid reservoirs while under pressure (up to 600 atm) are demonstrated. Next, the system is used for the time-gated, spectral monitoring of endogenous NADH fluorescence under pressure. Spectrofluorimetric measurements utilize a nitrogen-discharge laser for sub-nanosecond pulse width, 337-nm wavelength excitation, and an intensified CCD coupled to a spectrograph for nanosecond-gated spectral detection. Because emission from NADH is routinely used at ambient pressure for monitoring mitochondrial function, we validate the system by observing the pressurized response of Saccharomyces cerevisiae (baker's yeast) to mitochondrial functional modifiers (e.g., cyanide). Because the system is compatible with both spectroscopy and sub-cellular resolution microscopy imaging, the system represents a robust tool for investigating the biophysical effects of pressure on cellular systems.