In the last years, many devices dedicated to high content (HC) microscopy (imaging or screening) have been developed. These systems allow to measure in a fast, automated and reproducible way a large number of biological parameters, whether we work on thin (e.g. 2D cell culture) or thick samples (e.g. 3D cell culture). The main goal of this workshop is to give the necessary tools to improve 3D acquisitions in a HCS context. From samples' preparation to instrumental calibration, we will present all the key points to the successful implementation of a 3D HCS. We will use as a 3D model spheroid of approximately 200µm in size that were obtained from a pancreatic tumor cell line. Our workshop will address and illustrate efficiently the following issues: A. On sample preparation, how to choose the: (i) Fluorescent probes and immunolabeling techniques, (ii) Clearing medium, and (iii) Mounting/Support. B. On the instrumental calibration: (i) How to choose the objective (e.g. multi-immersion) accordingly to the refractive index of sample mounting medium (ii) The relevance of the disk (spinning) to improve speed, depth, and resolution. Finally, we will also discuss the various existing softwares available for imaging processing. Being able, for the different steps, to rethink what to do, or rather to avoid “stupid errors”, will be put forward. The participants will be actors and feel free to come and modify, propose an alternative approach/parameters on the machine that can improve the result. At the end of the workshop, participants will acquire theoretical and practical knowledge to perform sample preparation (including clearing approaches and immunolabeling strategies) on spheroids and that will allow them to improve their 3D HCS experiments by making better choices on 3D sample preparation, HCS acquisition, and analysis.