Time-resolved action spectroscopy together with a fs-pump probe scheme is used in an electrostatic ion-storage ring to address lifetimes of specific vibrational levels in electronically excited states. Here we specifically consider the excited-state lifetime of cryogenically cooled green fluorescent protein (GFP) chromophore anions which is systematically measured across the S0-S1 spectral region (450-482 nm). A long lifetime of 5.2 ± 0.3 ns is measured at the S0-S1 band origin. When exciting higher vibrational levels in S1, the lifetime changes dramatically. It decreases by more than two orders of magnitude in a narrow energy region ∼250 cm-1 (31 meV) above the 0-0 transition. This is attributed to the opening of internal conversion over an excited-state energy barrier. The applied experimental technique provides a new way to uncover even small energy barriers, which are crucial for excited-state dynamics.