As the closest living relatives of animals, choanoflagellates have brought crucial information to reconstruct the evolutionary origin of animals. Notably, certain choanoflagellate species can engage in facultative multicellular development resembling the early stages of embryogenesis. In the past few years, Salpingoeca rosetta has emerged as a tractable model for choanoflagellate cell biology and multicellular development, notably through mutant screens and CRISPR/Cas9-mediated gene knockout (KO). However, existing KO pipelines have variable and sometimes low efficiency, frequently requiring isolation and genotyping of hundreds of clones without guarantee to obtain a KO strain. Here, we present a robust method for gene inactivation in S. rosetta that relies on insertion by CRISPR/Cas9 of a single 1.9 kb cassette encoding both a premature termination sequence and an antibiotic resistance gene. We show that this approach allows robust, fast and efficient isolation of KO clones after antibiotic selection. As a proof of principle, we first knocked out all three genes previously proposed to regulate S. rosetta multicellular development in a published mutant screen (rosetteless, couscous and jumble), and confirmed all three KOs abolished multicellular development. Whole genome sequencing revealed a unique specific insertion of the termination/resistance cassette in KO strains. To showcase the potential of this method for de novo characterization of candidate developmental genes, we then inactivated three genes encoding homologs of components of the Hippo pathway, which controls cell proliferation and multicellular size in animals: hippo, warts and yorkie. Interestingly, warts KO rosettes were consistently about twice larger than their wild-type counterparts, indicating that our KO pipeline has the potential to rapidly reveal novel loss-of-function phenotypes of biological interest. We propose that this method has the potential to accelerate choanoflagellate functional genetics.