The octamer-binding transcription factor 4 (Oct4) is essential for maintaining the self-renewal and pluripotency of embryonic stem cells (ESCs). Post-translational modifications (PTMs) of Oct4 critically control its structure, function and intracellular localization. However, determination of Oct4 PTM profiles has largely been restricted by the quantity and purity of the Oct4 protein samples required for mass spectrometric analyses. In this study, by incubating the Escherichia coli-derived His-tagged Oct4 proteins with the whole cell lysates of a variety of human cells followed by retrieving the reacted Oct4 proteins with the Ni-NTA beads, we developed a labor- and cost-effective in vitro PTM method that allowed for mass spectrometric determination of the phosphorylation profiles of Oct4 proteins exposed to various cell-free systems. A number of Oct4 phosphorylation sites that were commonly present in all the cell-free systems or specifically present in a particular cellular context were identified, indicating that Oct4 is controlled by both common and distinct PTM regulatory pathways. Our work provided a proof-of-concept that such a cell-free system-based in vitro PTM approach can be applied to systematically map out the physiologically-relevant PTM sites in Oct4 proteins, which opened up an avenue to fully decipher the Oct4 PTM barcodes in various cellular contexts. Copyright 2014 The Authors. Published by Elsevier Inc. All rights reserved.