A simple, scalable, and fast procedure for the isolation of Chlamydomonas flagella is described. Chlamydomonas can be synchronously deflagellated by treatment with chemicals, pH shock, or mechanical shear. The Basic Protocol describes the procedure for flagellar isolation using dibucaine to induce flagellar abscission; we also describe the pH shock method as an Alternate Protocol when flagellar regeneration is desirable. Sub-fractionation of the isolated flagella into axonemes and the membrane + matrix fraction is described in a Support Protocol.