The study introduces One-Tip, a streamlined proteomics workflow that integrates efficient sample preparation with precise data-independent acquisition (nDIA) analysis. This method simplifies sample processing, enabling the reproducible identification of over 9000 proteins from ~1000 HeLa cells. It also identifies ~6000 proteins in single cells from early mouse embryos and over 3000 proteins per HeLa cell using the Uno Single Cell Dispenser. One-Tip is extended to analyze extracellular vesicles (EVs) extracted from blood plasma, identifying over 3000 proteins from 16 ng of EV preparation. The workflow is designed to overcome challenges in bulk and single-cell proteomics, offering high depth and throughput across various sample types. One-Tip requires only two pipetting steps and utilizes a commercially available Evotip™ for cell lysis and digestion. After an hour-long incubation, samples are ready for LC-MS/MS analysis. The workflow includes a surfactant for efficient cell membrane lysis and protein solubilization without denaturation. It minimizes sample handling, ensuring high proteome coverage and reducing the need for sonication and buffer exchanges. The workflow is efficient, scalable, and compatible with automated systems, enabling thousands of samples to be processed daily. The study demonstrates that One-Tip achieves comparable proteome coverage and quantification precision to the Protein Aggregation Capture (PAC) method, even with a low number of cells. It successfully analyzes single-cell proteomes of mouse pre-implantation embryos, identifying over 6216 protein groups across different developmental stages. The workflow also enables the analysis of extracellular vesicles from blood plasma, identifying over 3000 proteins from 16 ng of EV preparation. One-Tip is shown to be applicable to non-cellular samples, such as EVs, and is compatible with various platforms, including the Uno Single Cell Dispenser. The study highlights the versatility and sensitivity of One-Tip, demonstrating its potential to transform proteomics by providing a simple, efficient, and high-throughput workflow. It enables comprehensive proteome coverage in minimal cells and single zygotes, offering a valuable tool for a wide range of proteomic applications. The workflow is adaptable to various sample types, including FACS-sorted cells, human pre-implantation embryos, rare cell populations, and tissue slices. The study also addresses the challenges of sample preparation in proteomics, offering a solution that is user-friendly, scalable, and suitable for a variety of experimental setups.The study introduces One-Tip, a streamlined proteomics workflow that integrates efficient sample preparation with precise data-independent acquisition (nDIA) analysis. This method simplifies sample processing, enabling the reproducible identification of over 9000 proteins from ~1000 HeLa cells. It also identifies ~6000 proteins in single cells from early mouse embryos and over 3000 proteins per HeLa cell using the Uno Single Cell Dispenser. One-Tip is extended to analyze extracellular vesicles (EVs) extracted from blood plasma, identifying over 3000 proteins from 16 ng of EV preparation. The workflow is designed to overcome challenges in bulk and single-cell proteomics, offering high depth and throughput across various sample types. One-Tip requires only two pipetting steps and utilizes a commercially available Evotip™ for cell lysis and digestion. After an hour-long incubation, samples are ready for LC-MS/MS analysis. The workflow includes a surfactant for efficient cell membrane lysis and protein solubilization without denaturation. It minimizes sample handling, ensuring high proteome coverage and reducing the need for sonication and buffer exchanges. The workflow is efficient, scalable, and compatible with automated systems, enabling thousands of samples to be processed daily. The study demonstrates that One-Tip achieves comparable proteome coverage and quantification precision to the Protein Aggregation Capture (PAC) method, even with a low number of cells. It successfully analyzes single-cell proteomes of mouse pre-implantation embryos, identifying over 6216 protein groups across different developmental stages. The workflow also enables the analysis of extracellular vesicles from blood plasma, identifying over 3000 proteins from 16 ng of EV preparation. One-Tip is shown to be applicable to non-cellular samples, such as EVs, and is compatible with various platforms, including the Uno Single Cell Dispenser. The study highlights the versatility and sensitivity of One-Tip, demonstrating its potential to transform proteomics by providing a simple, efficient, and high-throughput workflow. It enables comprehensive proteome coverage in minimal cells and single zygotes, offering a valuable tool for a wide range of proteomic applications. The workflow is adaptable to various sample types, including FACS-sorted cells, human pre-implantation embryos, rare cell populations, and tissue slices. The study also addresses the challenges of sample preparation in proteomics, offering a solution that is user-friendly, scalable, and suitable for a variety of experimental setups.