The cellular actin cortex is the cytoskeletal structure primarily responsible for the control of animal cell shape and as such plays a central role in cell division, migration, and tissue morphogenesis. Due to the lack of experimental systems where the cortex can be investigated independently from other organelles, little is known about its composition, assembly, and homeostasis. Here, we describe novel tools to resolve the composition and regulation of the cortex. We report and validate a protocol for cortex purification based on the separation of cellular blebs. Mass spectrometry analysis of purified cortices provides a first extensive list of cortical components. To assess the function of identified proteins, we design an automated imaging assay for precise quantification of cortical actomyosin assembly dynamics. We show subtle changes in cortex assembly dynamics upon depletion of the identified cortical component profilin. Our widely applicable integrated method paves the way for systems-level investigations of the actomyosin cortex and its regulation during morphogenesis. © 2013 Wiley Periodicals, Inc.