Spatially mapping protein abundance in situ can offer important insights into molecular mechanisms and the physiological functions of protein complexes. This is typically achieved by combining super-resolution microscopy to image fluorescently-labeled protein locations with statistical estimators to retrieve abundances, where accuracy is strongly impacted by labeling efficiency. We introduce quantitative expansion microscopy (qExM) as a method to estimate endogenous protein abundance on ExM data, which offers improved antibody targeting through molecular decrowding. Using cryo-fixation, we preserved ultrastructure and enhanced labeling efficiency to improve accuracy in abundance estimations. We benchmark the effectiveness of qExM by quantifying the stoichiometry of well-characterized nuclear pore complex subunits, and find a mean percent error of 9.4%. We further apply qExM to investigate the abundance of mitochondrial respiratory chain complexes in functionally distinct organelle subpopulations, and of mitochondrial respiratory chain super-complexes in differentially activated human T-cells. qExM provides a robust methodological framework for quantifying endogenous protein abundance in expanded samples in situ. ### Competing Interest Statement The authors have declared no competing interest. Swiss National Science Foundation, https://ror.org/00yjd3n13, 310030_215737 International Human Frontier Science Program Organization, https://ror.org/02ebx7v45, RGP0038/2021 European Research Council, CoG 819823 Piko