Molecular Pixelation (MPX) is an innovative, optics-free method for spatial proteomics of single cells, utilizing antibody-oligonucleotide conjugates (AOCs) and DNA-based, nanometer-sized molecular pixels. This approach enables the high-throughput, multiplexed analysis of protein spatial organization in single cells without the need for fluorescence microscopy. MPX works by sequentially associating AOCs into local neighborhoods using unique molecular identifiers (UPIs) derived from DNA pixels, forming over 1,000 spatially connected zones per cell in 3D. DNA sequencing reads are then computationally arranged into spatial proteomics networks for 76 proteins, allowing for the identification of known and novel patterns of protein spatial organization on chemokine-stimulated T cells. The method has been validated using peripheral blood mononuclear cells (PBMCs) and has demonstrated the ability to generate single-cell data based on protein abundance. It also allows for the quantification of protein clustering or polarization and colocalization, providing insights into cellular states and processes. MPX has been applied to study the effects of therapeutic antibodies and chemokine stimulation on T cell dynamics, highlighting its potential in defining cell states by the spatial arrangement of proteins. The method's advantages include high multiplexing, throughput, and spatial resolution, making it a promising tool for advancing spatial proteomics research.Molecular Pixelation (MPX) is an innovative, optics-free method for spatial proteomics of single cells, utilizing antibody-oligonucleotide conjugates (AOCs) and DNA-based, nanometer-sized molecular pixels. This approach enables the high-throughput, multiplexed analysis of protein spatial organization in single cells without the need for fluorescence microscopy. MPX works by sequentially associating AOCs into local neighborhoods using unique molecular identifiers (UPIs) derived from DNA pixels, forming over 1,000 spatially connected zones per cell in 3D. DNA sequencing reads are then computationally arranged into spatial proteomics networks for 76 proteins, allowing for the identification of known and novel patterns of protein spatial organization on chemokine-stimulated T cells. The method has been validated using peripheral blood mononuclear cells (PBMCs) and has demonstrated the ability to generate single-cell data based on protein abundance. It also allows for the quantification of protein clustering or polarization and colocalization, providing insights into cellular states and processes. MPX has been applied to study the effects of therapeutic antibodies and chemokine stimulation on T cell dynamics, highlighting its potential in defining cell states by the spatial arrangement of proteins. The method's advantages include high multiplexing, throughput, and spatial resolution, making it a promising tool for advancing spatial proteomics research.