Mass spectrometry (MS)-based single-cell proteomics (SCP) enables proteome-wide analysis at single-cell resolution, offering insights into cellular heterogeneity, biological processes, and disease mechanisms. However, conventional nanoLC-MS workflows are constrained by long gradients and low throughput, limiting their application to small cohorts. Here, we present a multicolumn nanoLC–MS platform that achieves 5-minute separation windows with 100% duty cycles at ~100 nL/min, enabling the analysis of up to 288 single cells per day with minimal additional hardware. The system provides stable peptide separation, negligible carryover, and robust retention-time reproducibility across 1,200 consecutive injections. Over the course of this study, we successfully analyzed more than 4,000 samples at nearly 288 samples per day (SPD) throughput. The platform identified ~5,000proteins per 250 pg digest injection and an average of ~4,000 proteins per single HeLa cell with maxima exceeding 5,100, which is comparable to state-of-the-art longer-gradient workflows. Quantitative benchmarking with mixed-species standards confirmed the quantitative accuracy is largely maintained during extended acquisition . The workflow profiled 783 individual RAW264.7 macrophages cells exposed to LPS, enabling an unprecedented characterization of cellular heterogeneity within a uniform inflammatory stimulus. . Together, these results establish a robust and scalable platform for high-throughput SCP, demonstrating the feasibility of thousands of single-cell analyses within a single study while maintaining deep proteome coverage and biological interpretability.