There is a constant pressure to reduce timelines in mammalian cell line development (CLD) for biotherapeutic protein production. Demonstration of clonal derivation of the generated cell lines is key for health authorities' approval. To meet these regulatory and process-oriented demands, single-cell dispensers have become vital instruments for single-cell cloning. We conducted validation experiments with the UP.SIGHT (CYTENA GmbH) to determine this instrument's single-cell dispensing efficiency (SCDE) and probability of clonal derivation (p(clonal)). Process optimization to maximize clone recovery with several cell lines was also performed, focusing on cloning media and plate type. With a SCDE >97%, p(clonal) >99.99% and clone recovery values of up to 80%, the data reported here support the notion that the UP.SIGHT covers all steps in the single-cell dispensing process with assurance of clonality and colony tracking, leading to faster and more efficient CLD workflows. This work also serves as a guideline for instrument validation and guidance towards process optimization.