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Abstract Background With the prompt developments of regenerative medicine, the potential clinical applications of human embryonic stem cells have attracted intense attention. However, the labor-intensive and complex manual cell selection processes required during embryonic stem cell culturing have seriously limited large-scale production and broad applications. Thus, availability of a label-free, non-invasive platform to replace the current cumbersome manual selection has become a critical need. Results A non-invasive, label-free, and time-efficient optical platform for determining the quality of human embryonic stem cell colonies was developed by analyzing the scattering signals from those stem cell colonies. Additionally, confocal microscopy revealed that the cell colony morphology and surface structures were correlated with the resulting characteristic light scattering patterns. Standard immunostaining assay (Oct-4) was also utilized to validate the quality-determination from this light scattering protocol. The platform developed here can therefore provide identification accuracy of up to 87% for colony determination. Conclusions Our study here demonstrated that light scattering patterns can serve as a feasible alternative approach to replace conventional manual selection for human embryonic stem cell cultures.