Membrane fission manifests during cell division, synaptic transmission, vesicular transport, and organelle biogenesis, yet identifying proteins that catalyze fission remains a challenge. Using a facile and robust assay system of supported membrane tubes in a microscopic screen that directly monitors membrane tube scission, we detect robust GTP- and ATP-dependent as well as nucleotide-independent fission activity in the brain cytosol. Using previously established interacting partner proteins as bait for pulldowns, we attribute the GTP-dependent fission activity to dynamin. Biochemical fractionation followed by mass spectrometric analyses identifies the Eps15-homology domain-containing protein1 (EHD1) as a novel ATP-dependent membrane fission catalyst. Together, our approach establishes an experimental workflow for the discovery of novel membrane fission catalysts.