Culturing red blood cells (RBCs) in vitro presents a promising alternative to conventional blood donation, addressing challenges such as supply disruptions, donor variability, and limited availability of rare blood types. A critical step in this process is the efficient enucleation and subsequent maturation of erythroid cells. However, enucleation efficiency varies widely across cultures and remains poorly understood. Advancing the production of cultured RBCs requires deeper insight into the biological mechanisms that govern successful, and failed, enucleation. Given that enucleated cells lack active transcriptional programs, single-cell proteomics provides a powerful approach to dissect the protein-driven dynamics underlying this process. This project set out to characterize the temporal dynamics of erythroid cell maturation. Due to the heterogeneous nature of developing cell populations, single-cell approaches were essential for resolving distinct maturation stages. As maturing red blood cells are small, protein-poor, and dominated by haemoglobin (~90% of total protein), a key objective was to develop a method capable of analysing these minimal single-cell proteomes while distinguishing lower-abundance proteins from the overwhelming haemoglobin background. By supporting our industry collaborators in developing improved bioprocessing protocols for red blood cell culture, this project directly contributes to advancements in the manufacture of in vitro produced blood. The benefits extend well beyond our immediate partners: the ability to produce functional red blood cells at scale has far-reaching implications for healthcare systems worldwide. This includes enhancing the reliability and equity of blood supply, reducing dependency on donors, improving transfusion safety, and enabling access to rare blood types for patients with complex or uncommon needs. Ultimately, the project has the potential to benefit patients, clinicians, and health services globally by contributing to a more sustainable and controllable source of life-saving blood products. The successful generation of data in this project was made possible by the combined precision of the CellenONE single cell sorter and the high sensitivity and speed of the ThermoFisher Orbitrap ASTRAL mass spectrometer. The CellenONE enables gentle isolation and deposition of individual cells in nanolitre droplets, with image-based confirmation of each sorted event. These images provide valuable metadata, including cell diameter and morphology, which can be leveraged to validate classifications derived from mass spectrometry – such as distinguishing pyrenocytes from fully enucleated cells. The ability to rapidly acquire high-quality proteomic data from large numbers of single cells with inherently low protein content is currently unmatched, and is only feasible with the Orbitrap ASTRAL. When paired with IonOpticks chromatography columns, this platform represents the leading-edge solution for single-cell proteomics. Q-MAP was responsible for generating high-quality single-cell proteomics data across two erythroid cell types, capturing the critical transition through enucleation. This effort encompassed the full workflow which included precise cell sorting, optimized sample preparation, high-sensitivity proteomic acquisition, and large-scale spectral data processing. This project was undertaken in partnership with Red Cross Lifeblood and the ARC Centre of Excellence in Synthetic Biology. One of the most remarkable outcomes of the project was the ability for the workflow to identify and quantify hundreds of proteins in some of the smallest cells in the body, which largely consist of haemoglobin. The data quality allow for fine resolution of erythropoiesis.