Extracellular vesicles (EVs) are natural membranous vesicles with immense potential as drug delivery tools. However, their large-scale production remains a huge technical challenge, is time consuming, and expensive. Thus, EV mimetics (nanovesicles) generated from easily sourced red blood cells (RBCs) have gained vested interest as an effective and scalable drug delivery system. Their surface proteins (e.g., CD47) inherited from parental RBCs also improve their biocompatibility and bioavailability. Here, we outline a step-by-step guide for large-scale production of RBC nanovesicles using one-step extrusion method coupled to rapid density-cushion centrifugation. We also outline protocol for their extensive biophysical characterization (size and morphology using single particle analysis and cryogenic electron microscopy), and in-depth mass spectrometry-based proteome characterization. Finally, we outline two strategies (active loading during extrusion vs. passive loading via diffusion) to incorporate pharmacological compound(s) into nanovesicles and detect their loading using spectrophotometry.