A novel fluorescence lifetime imaging microscopy (FLIM) working with deep UV 240–280 nm wavelength excitations has been developed. UV-FLIM is used for measurement of defect-related fluorescence and its changes upon annealing from femtosecond laser-induced modifications in fused silica. This FLIM technique can be used with microfluidic and biosamples to characterize temporal characteristics of fluorescence upon UV excitation, a capability easily added to a standardmicroscope-based FLIM. UV-FLIMwas tested to show annealing of the defects induced by silica structuringwith ultrashort laser pulses. Frequency-domain fluorescencemeasurementswere converted into the time domain to extract long fluorescence lifetimes from defects in silica.