We present a simple strategy (integrated Multistep enzyme digestion, Enrichment, Database search, “iMEED”) to improve coverage of the phosphoproteome from less starting material that is faster than other commonly used approaches. iMEED has generated the most comprehensive phosphoproteome of murine microglia to date, comprising 8807 unique phosphopeptides in 36 LC-MS/MS runs from 300 ug of starting material.