Mass spectrometry has revolutionized cell signaling research by vastly simplifying the analysis of many thousands of phosphorylation sites in the human proteome. Defining the cellular response to perturbations in space and time is crucial for further illuminating functionality of the phosphoproteome. Here we describe µPhos (‘microPhos’), an accessible phosphoproteomics platform that permits phosphopeptide enrichment from 96-well cell culture experiments in <8 hours total processing time. By minimizing transfer steps and reducing liquid volumes to <100 µL, we demonstrate increased sensitivity, >90% selectivity, and excellent quantitative reproducibility. Employing highly sensitive trapped ion mobility mass spectrometry, we quantify >30,000 unique phosphopeptides in a human cancer cell line using 20 µg starting material, and confidently localize >10,000 phosphsites from 1 µg. This depth covers key signaling pathways, rendering sample-limited applications and perturbation experiments with hundreds of samples viable as we demonstrate by profiling the time-resolved response of leukemia cells to tyrosine kinase inhibitors.