A new optical-fiber-based spectrofluorometer for in vivo or in vitro detection of delayed fluorescence is presented and characterized. This compact setup is designed so that it can be readily adapted for future clinical use. Optical excitation is done with a nitrogen laser-pumped, tunable dye laser, emitting in the UV-vis part of the spectrum. Excitation and luminescence signals are carried to and from the biological tissues under investigation, located out of the setup enclosure, by a single optical fiber. These measurements, as well as measurements performed without a fiber on in vitro samples in a thermostable quartz cell, in a controlled-atmosphere enclosure, are possible due to the efficient collection of the laser-induced luminescence light which is collected and focused on the detector with a high aperture parabolic mirror. The detection is based on a gated photomultiplier which allows for time-resolved measurements of the delayed fluorescence intensity. Thus, relevant luminescence lifetimes, typically in the sub-microsecond-to-millisecond range, can be measured with near total rejection of the sample's prompt fluorescence. The instrument spectral and temporal resolution, as well as its sensitivity, is characterized and measurement examples are presented. The primary application foreseen for this setup is the monitoring and adjustment of the light dose delivered during photodynamic therapy. (C) 2011 Society of Photo-Optical Instrumentation Engineers (SPIE). [DOI: 10.1117/1.3558846]