The extraction and analysis of intact proteins from complex biological samples via femtosecond laser ablation are influenced by wavelength-dependent multiphoton and avalanche ionization processes. To investigate these effects, an intact protein mixture with molecular weights ranging from 9 kDa to 68 kDa as well as individual proteins were sampled using laser wavelengths spanning from the ultraviolet to the near-infrared. Our results suggest that visible and infrared wavelengths enable intact protein extraction, even for proteins with strong absorption in the visible spectrum. Ultraviolet wavelengths also yield intact extraction when long enough to avoid resonant absorption by the aromatic amino acids. Our findings support the hypothesis that minimizing multiphoton ionization helps preserve intact protein signals during femtosecond laser ablation. By isolating the role of laser wavelength, this study provides insight on high intensity laser–biomolecule interactions relevant to analytical techniques such as high-resolution mass spectrometry imaging.