We have performed nonlinear spectroscopic measurements to investigate the chemical structure/nonlinear optical property relations for a set of alkyl fluorene derivatives. The characterization method we have utilized is a femtosecond white-light continuum (WLC) pump-probe spectrometer that can rapidly characterize an organic sample’s nondegenerate two-photon absorption (2PA) spectrum. The nature of these experiments requires sophisticated data analysis. In particular, the relative group velocity mismatch between the pump and probe, which are at different frequencies, makes these pulses walk through each other within the thickness of the sample. For widely different frequencies, this can severely diminish the 2PA signal strength. However, given careful analysis, we have found good agreement with well-known semiconductor samples. Confidence in this method has allowed us to investigate the effects of solvism, electron-withdrawing character, conjugation length, and symmetry on the two-photon absorbing properties of these molecules. We have found an optimum solvent polarity as well as electron-withdrawing character which serves to maximize the strength of the 2PA in these materials. Different synthesis avenues have provided us with two different methods of extending the conjugation length that increases the nonlinearity as well. Finally, investigations of molecules with disparate symmetry have allowed us to identify the symmetry of the excited states. In addition, we present the first experimental study of the intermediate state resonance enhancement of nondegenerate 2PA in organic molecules. Using a simplified sum-over-states expression, we make comparisons between experiment and theory.