The microtubule (MT) cytoskeleton is required for many aspects of cell function, including the transport of intracellular materials, maintenance of cell polarity, and the regulation of mitosis. These functions are coordinated by MT-associated proteins (MAPs), which work in concert with each other, binding MTs and altering their properties. We have used an MT co-sedimentation assay, combined with 1D and 2D PAGE and mass spectrometry, to isolate MAPs from early Drosophila embryos. This technique has identified many novel proteins and an association with MTs for many known proteins, previously not described as associating with MTs.