Abstract: Under in situ conditions in biological tissues, the extraction, ionization, separation, and structural analysis of proteins remain in an exploratory stage. To date, no analytical strategies have been established that can simultaneously achieve high sensitivity and high throughput under such conditions. Consequently, the analysis of proteins within complex tissue environments remains highly challenging, particularly for low-abundance proteins, protein complexes, and membrane proteins. By enabling the direct desorption and ionization of proteins from tissue sections, in situ mass spectrometry (MS) provides a unique approach for investigating both their spatial distribution and molecular structural characteristics. Currently, in situ MS analysis of tissue samples still encounters several technical bottlenecks, including low ionization efficiency, limited gas-phase separation capabilities, and restricted depth of sequence and structural characterization. These limitations collectively hinder the high-throughput detection of intact proteins and reduce the precision of structural characterization, ultimately compromising analytical resolution and confidence in protein identification. In comparison with conventional liquid chromatography-mass spectrometry (LC-MS) approaches, the recent integration of ion mobility spectrometry (IMS) with top-down MS has offered a promising strategy to overcome these limitations. This multidimensional analytical framework demonstrates significant potential for enhancing protein detection throughput, improving spatial resolution in tissue analyses, and enabling more precise characterization of protein conformations and post-translational modifications, while preserving spatial integrity. This review provided a systematic overview of recent advances in in situ MS for protein ionization and spatial characterization within tissue samples. It specifically focused on critical challenges in protein extraction, in situ ionization, gas-phase separation, and structural analysis, discussing current technical bottlenecks and methodological considerations. Furthermore, the review explored the prospects of integrating IMS with top-down MS in in situ workflows, discussing how this combination could augment analytical capabilities and enable the spatially resolved characterization of intact proteins. The purpose of this review was to promote the development of spatial proteomics under in situ conditions, emphasizing high spatial resolution, high mass resolution, and precise structural characterization. By systematically summarizing current methodologies and technical challenges, this review aims to provide conceptual guidance for advancing in situ MS approaches that enable the analysis of intact proteins in tissue samples across a wide range of abundances, facilitating more accurate structural characterization and yielding clear, well-defined spatial information. Ultimately, these insights are intended to support the future development of comprehensive spatial proteomics workflows.