We present in this work a new technique, which combines laser photofragmentation spectroscopy with tandem mass spectrometry, for structural investigations of biomolecular ions in the gas phase. A novel apparatus was designed and built for the implementation of these studies. In this instrument closed shell ions are produced in the gas phase by electrospray ionization and stored in a hexapole ion trap prior to mass selection of a parent beam in a first quadrupole and then irradiation in an octupole ion guide using laser light. Mass analysis of the photofragmentaion products in a final stage quadrupole as a function of the wavelength generates an action spectrum. We applied this new technique to follow the microsolvation of charged amino acids in the gas phase. In these studies, the nanoelectrospray ionization source generates a distribution of water clusters of charged amino acids at various hydration levels. A particular size of cluster is selected and irradiated by infrared laser pulses resulting in the dissociation of one water molecule. Detection of the photofragmentation of this weak non-covalent bond allows us to generate the vibrational action spectrum of this particular cluster. We use a two-stage difference frequency mixing setup to produce laser light in the 2900–3800 cm-1, allowing us to probe the light-atom stretching region. IR photofragmentation spectra have been obtained for the hydrates of protonated and lithiated valine and those of protonated tryptophan. By probing the region of free and hydrogen-bonded N-H and O-H stretch vibrations and with the help of density functional theory calculations at the B3LYP/6-31++G** level, we relate spectral changes to the structure of clusters. In the study of lithiated valine water clusters, we addressed the question of zwitterion formation upon the combined effect of water and an external ion. Our data indicate the presence of the non-zwitterionic form of valine upon addition of up to four water molecules. In all of the species studied here, the hydration process is driven by solvation of the charge, and upon completion of a first shell around it, water preferentially forms a second solvation shell with no strong competition from other hydration sites on the amino acid backbone. Strikingly, similar water network structures have been observed at the highest hydration level of completely different species, probably indicating the existence of stable ordered water structures. We obtained evidence for a structrual change of the valine backbone in lithiated valine upon addition of the third water molecule, while no conformational change has been identified in the clusters of the protonated species. We have thus been able to answer questions related to the conformational preferences of the amino acid and the structuring of the water network.