Figure compares the extracted evolution-associated difference spectra for the first two components with the calculated excited-state absorption spectra of the S1(nπ*) state and with a linear combination of the absorption spectra for the two T1(ππ*) minima obtained from the calculations. Based on these results, the transient signal that appears around time zero in Figure can be confidently assigned to excited state absorption from the S1(nπ*) state, while the subsequent longer-lived transient species can be satisfactorily assigned to a linear combination of the absorption spectra of both T1(ππ*) minima. Furthermore, the spectrum of this long-lived transient species closely resembles the one reported for 6MP on the microsecond time scale, which has been assigned to the absorption spectrum of the T1 state. Hence, using the singlet depletion and relative actinometry methods, the triplet yield of 6MP was determined in this study to be 1.0 ± 0.2 at both pH 5.5 and 7.4 in PBS, relative to 6-thioguanosine. The triplet yield was not obtained for 6MP in pH 9.8 because no clear bleaching signal can be observed due to the blue shifted absorption spectrum (Figure ) of 6MP at pH 9.8. However, based on the similarities of the transient spectra regardless of the pH used (Figure ), a near-unity triplet yield is also expected for 6MP at pH 9.8. The triplet yield reported in this study is in good agreement with a triplet yield of 0.99 that was measured previously for 6MP in tetrahydrofuran using an energy transfer method. The efficient population of the triplet state is also consistent with the room-temperature phosphorescence reported in Figures S1 to S3 for 6MP. Hence, the transient spectra reported in Figure evidence that the T1 state is populated on an ultrafast time scale (i.e., within τ1), which is further supported by the vertical excitation energies and the SOCs reported in Tables and.