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Unravelling the cellular and molecular mechanisms underlying lung injury and repair requires precise spatial context. Profiling cell-to-cell transcriptional variability and spatial orientation has become increasingly sophisticated, but validating results at the protein level still remains challenging, particularly for low-expressed proteins or small-scale samples. Here, we present a workflow established by our group for spatial protein analysis in the lung by combining two commercially available platforms: (1) laser-assisted microdissection (LMD) with (2) a capillary electrophoretic-based immunoassay (CEI). Using this workflow, we demonstrate a simple, accessible, and sensitive method for spatially capturing regions of interest to investigate small-scale samples or low-expressed proteins. This workflow provides an additional option for orthogonal validation for researchers using omics-based approaches. Furthermore, we validated transcriptome analysis results at the protein level by applying this workflow to a pre-clinical model of cigarette smoke (CS)-induced lung injury. In line with the previous findings, the results showed a significant downregulation of the endothelial cell marker in LMD-enriched alveolar regions, suggesting spatial capillary rarefaction, and activation of the mitogen-activated protein kinase (MAPK) signalling pathway in pulmonary vasculature of CS-exposed mice. Our approach overcomes traditional challenges and provides new opportunities for understanding complex disease pathomechanisms and identifying potential therapeutic targets.