Femtosecond laser-induced ionization/dissociation (fs-LID) has been demonstrated as a novel ion activation method for use in tandem mass spectrometry. The technique opens the door to unique structural information about biomolecular samples that is not easily accessed by traditional means. fs-LID is able to cleave strong bonds while keeping weaker bonds intact. This feature has been found to be particularly useful for the mapping of post-translational modifications such as phosphorylation， which is difficult to achieve by conventional proteomic studies. Here we investigate the laser-ion interaction on a fundamental level through the characterization of fs-LID spectra for the protonated amino acids and two series of derivatized samples. The findings are used to better understand the fs-LID spectra of synthetic peptides. This is accomplished by exploring the effects of several single-residue substitutions.