Trypanosoma cruzi metacyclogenesis is a natural process that occurs inside the triatomine vector and corresponds to the differentiation of non-infective epimastigotes into infective metacyclic trypomastigotes. The biochemical alterations necessary for the differentiation process have been widely studied with a focus on adhesion and nutritional stress. Here, using a mass spectrometry approach, a large-scale phospho(proteome) study was performed with the aim of understanding the metacyclogenesis processes in a quantitative manner. The results indicate that major modulations in the phospho(proteome) occur under nutritional stress and after 12 and 24 h of adhesion. Significant changes involve key cellular processes, such as translation, oxidative stress, and the metabolism of macromolecules, including proteins, lipids, and carbohydrates. Analysis of the signalling triggered by kinases and phosphatases from 7,336 identified phosphorylation sites demonstrates that 260 of these sites are modulated throughout the differentiation process, and some of these modulated proteins have previously been identified as drug targets in trypanosomiasis treatment. To the best of our knowledge, this study provides the first quantitative results highlighting the modulation of phosphorylation sites during metacyclogenesis and the greater coverage of the proteome to the parasite during this process. The data are available via ProteomeXchange with identifier number PXD006171.