Ultrastructure Expansion Microscopy (U-ExM) is a recently developed technique that enables the increase of the spatial resolution within a cell or a tissue for microscopic imaging by physically expanding the sample. I report, for the first time, a detailed protocol validating the use of U-ExM in Trypanosoma cruzi and quantifying the expansion factors of different subcellular compartments. I was able to determine the localization patterns of different tubulin isoforms, such as alpha-tubulin and

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... eta-tubulin. Also, I immunolocalized acetylated and tyrosinated alpha-tubulin isotypes in epimastigotes and use mitochondrial cell-permeable dyes to identify this organelle. Finally, U-ExM was also performed in trypomastigotes and amastigotes validating this technique in all life cycle stages of T. cruzi.