Data-independent acquisition (DIA) coupled with label-free quantification (LFQ) has emerged as a powerful tool for quantitative proteomics. The sensitivity and reproducibility of DIA analysis for proteomics make it attractive for large-scale biological investigations. Here, we investigate a robust and reproducible DIA workflow on the Orbitrap Ascend Tribrid MS for accurately quantifying and identifying hundreds to thousands of proteins from single cell line to complex sample mixtures with a high background of human peptides. The DIA workflow investigated in the present study was operated by directly injecting samples onto a 50-cm µPAC Neo HPLC column, and the peptides were resolved in a 60-min, 30-min and an ultra-high throughput 9-min gradient operated by a Vanquish Neo UHPLC system. The eluted peptides were analyzed on an Orbitrap Ascend Tribrid MS operated in DIA mode. With the 30-minute active gradient, 6,700+ proteins and 45,000+ peptides were identified, along with a protein group CV of approximately 5%, suggesting that the 30-minute method provides the perfect amount of time to achieve throughput while maximizing identification and quantitative performance. To test the reliability of quantitation accuracy, we used two samples with different amounts of spiked microbial proteins to mimic biological samples where proteins might be up- or down-regulated under different conditions. The DIA workflow used for relative quantitation of E.coli and Yeast proteins in a high amount of human peptides as background yielded excellent quantitation accuracy across all ratios, with median values close to the theoretical ratios. We also observed A narrow distribution of all data points around the median values, indicating high quantitative accuracy and precision in the workflow. Altogether, our results demonstrate the excellent proteome coverage and quantitation performance of Orbitrap Ascend Tribrid MS in DIA analysis.