It is shown that absolute two-photon fluorescence excitation (TPE) cross-sections can be obtained without prior knowledge of the temporal profile of the excitation pulse by measuring the fluorescence generated using two spatially superimposed but temporally shifted halves of the excitation beam as a function of their relative delay. Measurements employed a Michelson interferometer with a mode-locked Ti:sapphire laser as the excitation source. The TPE cross-section for fluorescein ( ) obtained at 782 nm with this technique agrees with single-mode CW Ti:sapphire excitation data. We find no indication for a deviation from the power-squared dependence of TPE.