A fast and automated approach to measuring two-photon fluorescence excitation (TPE) spectra of fluor- ophores with high resolution (∼2 nm) by pulse shaping ultrabroad-bandwidth femtosecond laser pulses is demonstrated. Selective excitation in the range of 675-990 nm was achieved by imposing a series of specially designed phase and amplitude masks on the excitation pulses using a pulse shaper. The method eliminates the need for laser tuning and is, thus, suitable for non-laser-expert use. The TPE spectrum of Fluorescein was compared with independent measurements and the spectra of the pH-sensitive dye 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) in acidic and basic environments were measured for the first time using this approach. © 2010 Optical Society of America.