Abstract Monoclonalization refers to the isolation and expansion of a single cell derived from a cultured population. This is a valuable step in cell culture so as to minimize a cell line’s technical variability downstream of cell-altering events, such as reprogramming or gene editing, as well as for processes such as monoclonal antibody development. However, traditional methods for verifying clonality do not scale well, posing a critical obstacle to studies involving large cohorts. Without automated, standardized methods for assessing clonality post-hoc , methods involving monoclonalization cannot be reliably upscaled without exacerbating the technical variability of cell lines. We report the design of a deep learning workflow that automatically detects colony presence and identifies clonality from cellular imaging. The workflow, termed Monoqlo, integrates multiple convolutional neural networks and, critically, leverages the chronological directionality of the cell culturing process. Our algorithm design provides a fully scalable, highly interpretable framework, capable of analyzing industrial data volumes in under an hour using commodity hardware. In the present study, we focus on monoclonalization of human induced pluripotent stem cells (HiPSCs) as a case example. Monoqlo standardizes the monoclonalization process, enabling colony selection protocols to be infinitely upscaled while minimizing technical variability.