Purpose. To develop a protocol for MALDI (matrix-assisted laser desorption ionization) imaging mass spectrometry for mapping the distributions of α-crystallin and its modified forms in human lens tissue as a function of lens age and cataract. methods. Frozen human lenses were cryosectioned equatorially and axially into 20-μm-thick sections, and the sections were mounted onto conductive glass slides by methanol soft-landing. An ethanol washing procedure facilitated uniform matrix crystal formation by a two-step matrix deposition procedure to produce high-quality mass spectral data. Molecular images of modified and unmodified α-crystallin subunits were obtained from mass spectral data acquired in 100-μm steps across normal and cataractous lens sections. Proteins extracted from the lens sections were digested with endoproteinase Glu-C and subjected to mass spectrometric analysis for identification of modifications. results. Intact α-crystallin signals were detected primarily in the outer cortical fiber cells in lenses up to 29 years of age. Multiple truncation products were observed for α-crystallin that increased in abundance, both with distance into the lens and with lens age. Phosphorylated αB-crystallin forms were most abundant in the cortical region of older lenses. In axial sections, no significant anterior–posterior pole variation was observed. A previously unreported αA-crystallin mutation was detected in an age-matched cataractous human lens. conclusions. A method has been developed to spatially map the age-related changes of human lens α-crystallin by MALDI imaging mass spectrometry including a novel L52F αA-crystallin mutation in a cataractous lens. Application of this spatially resolved proteomic technique to lens biology enhances the understanding of α-crystallin protein processing in aging and diseased human lenses.