(A) Time-resolved fluorescence waveforms from RyR-targeted, FRET-based HTS of NCC library (10 μM test-compounds) performed in 384-well format. (B) The lifetime measurement affords ~30× higher precision than intensity detection. (C) The relationship between the HTS assay quality, Z′, and the signal window, ΔE, indicates that fluorescence lifetime enables excellent quality even for minuscule ΔE (of ≥0.03), relative to the fluorescence intensity, which requires a much larger ΔE (of >0.6) for the same quality. All fluorescent compounds that interfere with donor probe lifetime were removed from the data set.