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Summary: H2 production from green-microalgae, for energy purposes, is the ultimate goal of large-scale production. Here, we present a two-phase protocol for hydrogen production assay under ambient conditions using Chlamydomonas reinhardtii, which eliminates steps used previously, including centrifugation and resuspension with sulfur-deprived media. We detail steps for Chlamydomonas reinhardtii culture, acetate supply replenishment, anaerobic induction, and H2 quantification. This protocol enables large-scale experiments in an easy and cost-effective method while maintaining cells vital, crucial factors for transition to industrial scales.For complete details on the use and execution of this protocol, please refer to Elman et al. (2022). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.