We present a technique for fast wide-field fluorescence lifetime (FLT) imaging using simultaneous steady-state and time- (or frequency-) domain measurements acquired in a single shot and exploiting the theoretical dependence of fluorescence intensity on FLT. Using theory, simulations, and experiments, we show that the single-shot FLT (S-FLT) method can provide absolute FLTs at a higher signal-to-noise ratio compared to other high-speed FLT imaging techniques, without the need for sample-dependent system calibration or model training. We demonstrate that S-FLT can provide accurate tumor delineation in real-time during surgical resections in preclinical tumor models and in clinical patient specimens . We also extend S-FLT for real-time multiplexing and demonstrate this technique for quantifying dynamic FLT changes and for multiplexing of organ-targeted NIR fluorophores . Our results suggest that S-FLT can enable real-time quantitative high-throughput studies in preclinical applications and accurate tumor margin delineation in real-time during surgeries.