The number of scientists working with human stem cells has exponentially increased since the derivation of iPSCs; creating an urgent need to develop and adopt universal protocols for derivation and expansion of stem cell lines. Existing human ESC and iPSC lines have been created and propagated using a wide variety of manual and enzymatic methods creating extensive variability. Manual picking of stem cell colonies is the most common technique for derivation; however, this approach is unpractical for efficient, large-scale derivation due to the inconsistency of output associated with varying expertise, risk of contamination, high labor cost, and inability to effectively automate. An approach for automated isolation of individual stem cell colonies from surrounding cells for the purpose of creating new stem cell lines has been developed. Brightfield and fluorescent imaging was combined with laser-mediated isolation to provide more rapid derivation of new iPSC lines. Using an optical manipulation, all handling is performed within a sterile, closed environment. Results show that laser-mediated colony purification can be used to efficiently generate iPSC lines from human fibroblasts in a higher throughput manner. In addition, data show that laser-mediated iPSC derivation is more efficient and reproducible than manual colony picking methods. After isolation, iPSC colonies were expanded and then propagated by laser-mediated sectioning creating iPSC lines (free of manual and enzymatic disruption) that had normal stem cell morphology and expressed high levels of stem cell-associated genes/proteins. aCGH analyses demonstrated that iPSC lines propagated by laser-mediated passage were genetically stable after >6 months of culture. The use of laser-mediated colony purification combined with laser-mediated propagation significantly reduces the expertise, labor, and time associated with stem cell maintenance and provides a standardized protocol for efficient derivation and production of GMP-quality stem cell lines.