Through optimization of the printing process and orientation, a suitably developed surface area has been realized upon a 3D printed polymer substrate to facilitate chromatographic separations in a planar configuration. Using an Objet Eden 260VS 3D printer, polymer thin layer chromatography platforms were directly fabricated without any additional surface functionalization and successfully applied to the separation of various dye and protein mixtures. The print material was characterized using gas chromatography coupled to mass spectrometry and spectroscopic techniques such as infrared and Raman. Preliminary studies included the separation of colored dyes, whereby the separation performance could be visualized optically. Subsequent separations were achieved using fluorescent dyes and fluorescently tagged proteins. The separation of proteins was affected by differences in the isoelectric point (pI) and the ion exchange properties of the printed substrate. The simple chromatographic separations are the first achieved using an unmodified 3D printed stationary phase.