Chlamydomonas reinhardtii is a model organism for various processes, from photosynthesis to cilia biogenesis, and a great chassis to learn more about biofuel production. This is due to the width of molecular tools available, which have recently expanded with the development of a modular cloning system but, most importantly, with CRISPR/Cas9 editing now being possible. This technique has proven to be more efficient in the absence of a cell wall by using specific mutants or by digesting Chlamydomonas cell wall using the mating-specific metalloprotease autolysin (also called gametolysin). Multiple protocols have been used and shared for autolysin production from Chlamydomonas cells; however, they provide very inconsistent results, which hinders the capacity to routinely perform CRISPR mutagenesis. Here, we propose a simple protocol for autolysin production requiring transfer of cells from plates into a dense liquid suspension, gametogenesis by overnight incubation before mixing of gametes, and enzyme harvesting after 2 h. This protocol has shown to be highly efficient for autolysin production regardless of precise control over cell density at any step. Requiring a minimal amount of labor, it will provide a simple, ready-to-go approach to produce an enzyme critical for the generation of targeted mutants.