Live cell imaging is an established method that can be used to study keratinocyte division kinetics in vitro. To minimize the influence of the culture environment, unpassaged human keratinocytes can be utilized. To study the kinetics of single cells, keratinocytes are grown at clonal density and divisions are recorded utilizing time lapse photography. From this, lineage trees are constructed, allowing determination of cell cycle duration and division fates of individual keratinocytes. Here we describe a method for the culture of human keratinocytes for live cell imaging, methods for analysis of the photography, and how to assess proliferation, differentiation, and cell cycle duration.