Background/purposes: Understanding the two-photon excitation spectral characteristics and microscopic morphology of cutaneous collagen and elastic tissue components is important for applying multiphoton microscopy (MPM) in basic skin biology research and for clinical diagnosis. Methods: We developed a system for two-photon excitation spectral measurements at various excitation wavelengths. The microscopic morphology was studied using a commercial multiphoton microscope. Results: We obtained two-photon excitation fluorescence (TPEF) excitation–emission matrices (EEM), for the first time, of purified collagen and elastin samples, as well as in situ collagen and elastic fibers within excised human dermis. The EEM of the dermis was found to be similar to that of elastin. The excitation spectra for second harmonic generation (SHG) from purified collagen and excised dermis were also studied and were found to have similar spectral shapes. Conclusion: This study, using the EEM spectroscopic approach, confirmed a previous imaging study inference that in the dermis, TPEF predominantly originates from elastic fibers, while SHG originates solely from collagen fibers. The EEM data and SHG excitation spectra obtained in this study can be used to guide the selection of excitation wavelengths for MPM applications in basic skin biology research and for clinical diagnosis.