AbstractConventional (2D) monolayer culture fails to recapitulate the complexity of native tissue and organs. Recent advances in three-dimensional (3D) in vitro models, in particular spheroids and organoids, offer unprecedented means to replicate human tissues, organs and diseases in a petri dish. Indeed, tumor spheroids display hallmarks of solid tumor such as multicellular composition, spatial architecture, diffusion barrier, differential gene expression and drug resistance, rendering them the model of choice in anti-tumor drug screenings. To date, preparation of spheroids for high throughput screenings are poised to rely on the random aggregation of a cell suspension in overlay culture labware (ca. non-adherent multi-well plates) which raises concern about sample homogeneity and cellular debris scattering. Moreover, to yield reproducible and predictive assays, pre-selection of spheroids based on their size, morphology and intern organization is critical. Hitherto, there is no tractable automated and high throughput means to isolate and manipulate spheroids. By merging Scienion’s sciDROP NANO® low-volume dispensing in air technology and cellenONE® software, Cellenion has developed the spheroONE, an innovative platform to isolate, inspect and dispense large-particle such as 3D spheroids. Here, we highlight the capabilities of the platform by demonstrating our ability to isolate spheroids with high accuracy (up to 100% single spheroid per well) and sort them according to user-defined characteristics (size and morphology). The preparation of assay-ready plates by isolating and dispensing spheroids prepared in bulk has the potential to significantly accelerate drug screening processes and improve their predictability.