This dataset contains proteomic analyses that validate the spatial proteomic technology, NicheProt, and its application to investigate dendritic cell phenotypes in inflammed mouse spleens. NicheProt is a 3D optical microscopy-guided, photobleaching-mediated cell barcoding approach to isolate intact cell types from specific microanatomical niches. Integrating with sequential bottom-up LC-MS/MS analysis we identified two distinct CD11c⁺ dendritic cell phenotypes defined by their spatial distribution. These compartment-specific populations displayed differential expression of 54 proteins. This approach enables cell-type and microregion-resolved proteomic insights, revealing previously unrecognized cell subtypes and their roles within distinct tissue compartments.