AbstractThe spatial organization of molecules in a cell is essential for their functions. While current methods focus on discerning tissue architecture, cell–cell interactions, and spatial expression patterns, they are limited to the multicellular scale. We present Bento, a Python toolkit that takes advantage of single-molecule information to enable spatial analysis at the subcellular scale. Bento ingests molecular coordinates and segmentation boundaries to perform three analyses: defining subcellular domains, annotating localization patterns, and quantifying gene–gene colocalization. We demonstrate MERFISH, seqFISH + , Molecular Cartography, and Xenium datasets. Bento is part of the open-source Scverse ecosystem, enabling integration with other single-cell analysis tools.