Reversible electroporation is a method to introduce molecules into cells by increasing the permeability of their membranes, thanks to the application of pulsed electric fields. One of its main biomedical applications is electro-chemotherapy, where electroporation is used to deliver anticancer drugs into tumor tissues. To improve our understanding of the electroporation effect on tissues and select efficient treatments, in vitro tumor models are needed. Cell spheroids are relevant models as they can reproduce tumor microenvironment and cell–cell interactions better than 2D cell cultures. Various methods offering a relatively simple workflow are now available for their production. However, electroporation protocols usually require handling steps that may damage spheroids and result in random spacing, inducing variations in electric field distribution around spheroids and non-reproducible electroporation conditions. In addition, only a few microsystems allow the production and electroporation of spheroids, and the spheroids produced lack reproducibility in size and location. To overcome these issues, we developed a unique device enabling culture, monitoring, and electroporation of hundreds of regular spheroids in parallel, with a design ensuring that all spheroids are submitted to the same electric field conditions. It is comprised of a microfluidic chamber encompassing a micro-structured agarose gel, allowing easy medium exchange while avoiding spheroid handling. It also enables optical imaging of spheroids in situ, thanks to transparent electrodes. In this paper, we describe the fabrication and characterization of the developed microsystem and demonstrate its applicability to electroporation of a network of spheroids. We present a first successful application as an anticancer drug testing platform, by evaluating the bleomycin effect on HT29 colorectal cancer cell spheroids. This work opens new perspectives in the development of in vitro assays for the preclinical evaluation of electroporation-based treatment.