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, dynamics and regulation of the cell cortex based on an integrative and multidisciplinary method encompassing laser ablation, high-speed confocal imaging, automated quantitative image analysis, scanning electron microscopy and mass spectrometry. We show that cellular blebs, spherical membrane protrusions that exhibit de novo actin cortex assembly, constitute a rare model system to study the actin cortex and form excellent purified cortical fractions when isolated. We report a first extensive parts list of cortical components and present 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 of cortex regulation during morphogenesis.