In recent years, life science research has experienced a significant shift, moving away from conducting bulk cell interrogation towards single-cell analysis. It is only through single-cell analysis that a complete understanding of cellular heterogeneity, and the interplay between various cell types that are fundamental to specific biological phenotypes, can be achieved. The hematopoietic system is a prime example of such a complex hierarchy, where Hematopoietic stem cells (HSC) are the origin of all cell lineages contained therein. Acute myeloid leukemia (AML), a perturbed state of hematopoiesis, is also hierarchically organized, with leukemia stem cells (LSC) at the apex. Successful eradication of AML will likely depend on specific targeting of these tumour-initiating cells, in turn requiring their molecular characterization. Currently, we are able to isolate LSC based on CD34 and CD38 expression levels of primary AML cells. These sorted fractions, however, vary in their levels of LSC, and thus should be considered LSC-enriched, rather than pure LSC. Consequently, this significant level of heterogeneity within the LSC-enriched fractions hampers the molecular characterization of true LSC. Here, we have taken a well-characterized primary AML patient sample and subjected it to fluorescence-activated cell sorting, combined with a newly developed single-cell proteomics strategy to identify the protein landscapes of individual cells within the LSC, progenitor and blast populations of this patient. By using the latest state-of-the-art LC-MS instrumentation with intelligent data acquisition, combined with the new TMTPro reagents, this has resulted in an unprecedented map of protein expression in individual AML cells. Furthermore, we developed a computational pipeline (SCeptre) that effectively normalizes the data, clusters the cells, integrates available FACS data (i.e immunophenotype) and permits the extraction of cell-specific proteins. We found a strong enrichment of stem cell specific proteins in the LSC and progenitor compartments compared to blasts, and the resulting protein signatures clearly distinguish the various differentiation stages that were isolated. The approach presented here lays a solid foundation for implementing global single-cell proteomics studies in proteomics labs across the world.