Abstract: We have developed a laser ablation sampling technique for matrix-assisted laser desorption ionization (MALDI) mass spectrometry and tandem mass spectrometry (MS/MS) analyses of in-situ digested tissue proteins. Infrared laser ablation was used to remove biomolecules from tissue sections for collection by vacuum capture and analysis by MALDI. Ablation and transfer of compounds from tissue removes biomolecules from the tissue and allows further analysis of the collected material to facilitate their identification. Laser ablated material was captured in a vacuum aspirated pipette-tip packed with C18 stationary phase and the captured material was dissolved, eluted, and analyzed by MALDI. Rat brain and lung tissue sections 10 μm thick were processed by in-situ trypsin digestion after lipid and salt removal. The tryptic peptides were ablated with a focused mid-infrared laser, vacuum captured, and eluted with an acetonitrile/water mixture. Eluted components were deposited on a MALDI target and mixed with matrix for mass spectrometry analysis. Initial experiments were conducted with peptide and protein standards for evaluation of transfer efficiency: a transfer efficiency of 16% was obtained using seven different standards. Laser ablation vacuum capture was applied to freshly digested tissue sections and compared with sections processed with conventional MALDI imaging. A greater signal intensity and lower background was observed in comparison with the conventional MALDI analysis. Tandem time-of-flight MALDI mass spectrometry was used for compound identification in the tissue. Graphical Abstract ᅟ.