We describe a high-throughput time-resolved fluorescence (TRF) spectrometer, able to detect multiple fluorescence lifetimes across 384 wells with short (< 5 min.) read times using direct waveform recording. The instrument combines high-energy pulsed laser sources (5-10 kHz repetition rate, 1-3 ns pulse width) with a photomultiplier and high-speed digitizer (1 GHz, effectively 5 GHz with interleaving) to record a complete fluorescence decay waveform after each pulse. Single-well measurements of dyes with 200-fold signal averaging (0.1 s acq. time) yield lifetimes comparable in accuracy and precision to single photon counting (SPC.) Integrated software enables immediate analysis by fitting exponential decays or by calculating a model-independent truncated first moment. In a 384-well format changes in quencher concentration are readily seen, with the first moment calculation providing resolution comparable to exponential decay models. Further, we are able to resolve relative mole fractions in two-dye mixtures when pure samples are used as standards. In multiple-well experiments the variation in total measured fluorescence is comparable to steady-state instruments, while the precision in lifetime is better than 2%. These features will enable high-throughput TRF experiments to detect changes to structure and dynamics in solution, cells and reconstituted systems. Acknowledgements: Spectroscopy experiments were performed at the Biophysical Spectroscopy Center, University of Minnesota. Excellent computational resources were provided by the Minnesota Supercomputing Institute. This work was funded by NIH grants to DDT (R01 AR32961, P30 AR057220) and KJP (T32 AR007612.)