The study introduces a novel data-independent acquisition (DIA) strategy called narrow-window DIA (nDIA), which combines high-resolution MS1 scans with parallel, ultra-fast MS/MS scans at a rate of ~200 Hz using 2-Th isolation windows. This approach, achieved by pairing a quadrupole Orbitrap mass spectrometer with the asymmetric track lossless (Astral) analyzer, enables comprehensive proteome profiling with high resolution, sensitivity, and mass accuracy. The nDIA strategy allows for profiling of >100 full yeast proteomes or 48 human proteomes per day, with a depth of ~10,000 human protein groups in half an hour or ~7,000 proteins in 5 minutes, representing 3× higher coverage compared to current state-of-the-art mass spectrometry. The method demonstrates high quantitative precision and accuracy in a three-species proteome mixture, quantifying over 14,000 protein groups in a single half-hour run. The study also introduces an optimized method for human proteome profiling, combining offline high-pH reversed-phase peptide fractionation and short online liquid chromatography gradients with nDIA, achieving nearly complete coverage of the human proteome within 3–4.5 hours. The nDIA strategy is further validated in systems biology studies, such as comprehensive yeast proteome profiling and clinical proteomics, demonstrating its potential for high-throughput and in-depth proteome analysis.The study introduces a novel data-independent acquisition (DIA) strategy called narrow-window DIA (nDIA), which combines high-resolution MS1 scans with parallel, ultra-fast MS/MS scans at a rate of ~200 Hz using 2-Th isolation windows. This approach, achieved by pairing a quadrupole Orbitrap mass spectrometer with the asymmetric track lossless (Astral) analyzer, enables comprehensive proteome profiling with high resolution, sensitivity, and mass accuracy. The nDIA strategy allows for profiling of >100 full yeast proteomes or 48 human proteomes per day, with a depth of ~10,000 human protein groups in half an hour or ~7,000 proteins in 5 minutes, representing 3× higher coverage compared to current state-of-the-art mass spectrometry. The method demonstrates high quantitative precision and accuracy in a three-species proteome mixture, quantifying over 14,000 protein groups in a single half-hour run. The study also introduces an optimized method for human proteome profiling, combining offline high-pH reversed-phase peptide fractionation and short online liquid chromatography gradients with nDIA, achieving nearly complete coverage of the human proteome within 3–4.5 hours. The nDIA strategy is further validated in systems biology studies, such as comprehensive yeast proteome profiling and clinical proteomics, demonstrating its potential for high-throughput and in-depth proteome analysis.