Multiphoton dissociation and ionization of 2,5-dihydroxyacetophenone (DHAP), an important matrix compound in UV matrix-assisted laser desorption/ionization (MALDI), is studied in a molecular beam at 355 nm using multimass ion imaging mass spectrometer and time-of-flight mass spectrometry. For laser fluence larger than 130 mJ/cm2, nearly all of the irradiated molecules absorb at least one photon. The absorption cross section was found to be σ = 1.3(±0.2) × 10-17cm 2. Molecules excited by two photons quickly dissociate into fragments. The major channels are (1) C6H3(OH) 2COCH3 →C6H3(OH)2CO + CH3 and (2) C6H3(OH)2COCH 3 → C6H3(OH)2 + COCH 3.. Molecules absorbing three or more photons become parent ions or crack into smaller ionic fragments. The concentration ratio of ions (parent ions and ionic fragments) to neutral fragments is about 10-6:1. Changing the molecular beam carrier gas from He at 250 Torr to Ar at 300 Torr results in molecular beam clustering (dimers and trimers). Multiphoton ionization of clusters by a 355 nm laser beam produces only dimer cations, (C 6H3(OH)2COCH3)2 +. Protonated clusters or negatively charged ions, observed from a solid sample of DHAP using 355 nm multiphoton ionization, were not found in the molecular beam. The experimental results indicate that the photoionization occurs in the gas phase after DHAP vaporizes from the solid phase may not play an important role in the MALDI process.