One-Tip is a novel proteomics methodology that integrates efficient, one-pot sample preparation with precise, narrow-window data-independent acquisition (nDIA) analysis. This approach simplifies sample processing, enabling the reproducible identification of >9000 proteins from ~1000 HeLa cells and ~6000 proteins from single cells in early mouse embryos. The versatility of One-Tip is further demonstrated by its application in single-cell proteomics using the Uno Single Cell Dispenser™, identifying >3000 proteins per HeLa cell. Additionally, One-Tip is extended to analyze extracellular vesicles (EVs) extracted from blood plasma, identifying >3000 proteins from 16 ng EV preparation. The workflow is designed to overcome the challenges of conventional mass spectrometry-based proteomics, such as sample losses, reproducibility issues, and high costs, making it more accessible and efficient for a wide range of sample types. The study highlights the superior analytical depth and throughput of One-Tip compared to standard workflows, making it a valuable tool for modern proteomics research.One-Tip is a novel proteomics methodology that integrates efficient, one-pot sample preparation with precise, narrow-window data-independent acquisition (nDIA) analysis. This approach simplifies sample processing, enabling the reproducible identification of >9000 proteins from ~1000 HeLa cells and ~6000 proteins from single cells in early mouse embryos. The versatility of One-Tip is further demonstrated by its application in single-cell proteomics using the Uno Single Cell Dispenser™, identifying >3000 proteins per HeLa cell. Additionally, One-Tip is extended to analyze extracellular vesicles (EVs) extracted from blood plasma, identifying >3000 proteins from 16 ng EV preparation. The workflow is designed to overcome the challenges of conventional mass spectrometry-based proteomics, such as sample losses, reproducibility issues, and high costs, making it more accessible and efficient for a wide range of sample types. The study highlights the superior analytical depth and throughput of One-Tip compared to standard workflows, making it a valuable tool for modern proteomics research.