Zeaxanthin (Zea) is a key component in the energy-dependent, rapidly reversible, nonphotochemical quenching process (qE) that regulates photosynthetic light harvesting. Previous transient absorption (TA) studies suggested that Zea can participate in direct quenching via chlorophyll (Chl) to Zea energy transfer. However, the contamination of intrinsic exciton–exciton annihilation (EEA) makes the assignment of TA signal ambiguous. In this study, we present EEA-free TA data using Nicotiana benthamiana thylakoid membranes, including the wild type and three NPQ mutants (npq1, npq4, and lut2)...