Generation of induced pluripotent stem (iPS) cells from somatic cells has been successfully achieved by ectopic expression of four transcription factors, Oct4, Sox2, Klf4 and c-Myc, also known as the Yamanaka factors. In practice, initial iPS colonies are picked based on their embryonic stem (ES) cell-like morphology, but often may go on to fail subsequent assays, such as the alkaline phosphate (AP) assay. In this study, we co-expressed through lenti-viral delivery the Yamanaka factors in amniotic fluid-derived (AF) cells. ES-like colonies were picked onto a traditional feeder layer and a high percentage AF-iPS with partial to no AP activity was found. Interestingly, we obtained an overwhelming majority of fully stained AP positive (AP+) AF-iPS colonies when colonies were first seeded on a feeder-free culture system, and then transferred to a feeder layer for expansion. Furthermore, colonies with no AP activity were not detected. This screening step decreased the variation seen between morphology and AP assay. We observed the AF-iPS colonies grown on the feeder layer with 28% AP+ colonies, 45% AP partially positive (AP+/-) colonies and 27% AP negative (AP-) colonies, while colonies screened by the feeder-free system were 84% AP+ colonies, 16% AP+/- colonies and no AP- colonies. The feeder-free screened AP+ AF-iPS colonies were also positive for pluripotent markers, OCT4, SOX2, NANOG, TRA-1-60, TRA-1-81, SSEA-3 and SSEA-4 as well as having differentiation abilities into three germ layers in vitro and in vivo. In this study, we report a simplistic, one-step method for selection of AP+ AF-iPS cells via feeder-free screening.