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Herein, these challenges are addressed by developing an oxygen\u2010tolerant photoinitiated polymerization\u2010induced self\u2010assembly (Photo\u2010PISA) process, allowing for the microliter\u2010scale (10\u00a0\u00b5L) synthesis of protein\u2010loaded polymersomes directly in 1536\u2010well plates. High\u2010resolution techniques capable of analysis at a single particle level are employed to analyze protein encapsulation and release mechanisms. Using confocal microscopy and super\u2010resolution stochastic optical reconstruction microscopy (STORM) imaging, their ability to deliver proteins into the cytosol following endosomal escape is subsequently visualized. Lastly, the adaptability of these polymersomes is exploited to encapsulate and deliver a prototype vaccine antigen, demonstrating its ability to activate antigen\u2010presenting cells and support antigen cross\u2010presentation for applications in subunit vaccines and cancer immunotherapy. This combination of ultralow volume synthesis and efficient intracellular delivery holds significant promise for unlocking the high throughput screening of a broad range of otherwise cost\u2010prohibitive or early\u2010stage therapeutic protein and vaccine antigen candidates that can be difficult to obtain in large quantities. 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