Rapid and comprehensive analysis of complex proteomes across large sample sets is essential to fulfill the potential of systems biology. We present a novel mass spectrometry (MS) method that integrates narrow-window data-independent acquisition (nDIA) with short-gradient micro-flow chromatography, enabling profiling of >240 samples per day. The optimized MS method identifies 6,201 and 7,466 human proteins with 1- and 2-min gradients, respectively.