Brain organoids are in vitro 3D cultures generated in the lab from human induced pluripotent stem cells or embryonic stem cells and can mimic the human brain structure and function. Specifically, they reproduce to some extent in vivo developmental events as they consist of diverse cell types, such as apical radial glial cells, intermediate progenitors, basal radial glial cells, and neurons forming stratified cortical layers similar to what is observed in the human brain in vivo. Due to cytoarchitecture similarities between the human brain and brain organoids, the latter have been proposed as excellent models for studying human brain development and disease. Thus, genome manipulation in brain organoids is crucial for investigating the functions of specific genes and mutations that have been associated with brain-related disorders. For this reason, gene manipulation has been implemented in brain organoids in the last few years. Here, we describe a step-by-step protocol for gene expression manipulation and analyses in brain organoids via acute electroporation that we have optimized based on the in vivo electroporation that has been widely used in animal models. This easy-to-apply protocol is fast and robust and facilitates the precise spatiotemporal manipulation of the expression of any gene of interest.