The absolute femtosecond pump-probe signal strength of deprotonated fluorescein in basic methanol is measured. Calculations of the absolute pump-probe signal based on the steady-state absorption and emission spectrum that use only independently measured experimental parameters are carried out. The calculation of the pump-probe signal strength assumes the pump and probe fields are both weak and includes the following factors: the transverse spatial profile of the laser beams; the pulse spectra; attenuation of the propagating pulses with depth in the sample; the anisotropic transition probability for polarized light; and time-dependent electronic population relaxation. After vibrational and solvent relaxation are complete, the calculation matches the measurement to within 10% error without any adjustable parameters. This demonstrates quantitative measurement of absolute excited state population.