Collagen fiber orientation plays an important role in many cell properties and actions in vivo. Collagen and other matrix proteins are aligned in many tissues during normal functioning. For example, cardiomyocytes align in the heart to produce a synchronously beating tissue. The extra-cellular matrix environment, including collagen, is aligned along the cells. This matrix helps with cell adhesion and the alignment of the fibers also contributes to the anisotropic mechanical property of the tissue. While it is easy to replicate randomly oriented collagen in vitro, it is much more difficult to create aligned collagen matrices for cell culture. In this work, a novel inkjet printer-based collagen alignment technique was established. A 1 mg/ml rat tail collagen type I solution was printed, using a modified HP DeskJet 500 printer, onto plasma cleaned and UV sterilized glass slides. The collagen was printed in an eight line pattern, designed in Microsoft Word with 87.5 μm by 23.1 mm lines. The pattern was printed three successive times on each slide to complete the alignment. Immunofluorescence imaging of primary antibodies specific to collagen type I indicated that the heat involved in the printing process was not great enough to denature the collagen. The extent of collagen alignment was quantified using atomic force microscopy and compared to random collagen films and collagen films aligned using a mechanical scraping method. Additionally, neonatal rat cardiomyocytes were cultured on the aligned matrices. These cells require extracellular matrix alignment to maintain their in vivo-like phenotype during in vitro culture. The cells grew along the lines of collagen and coordinated beating, indicating the success of the aligned matrix. This collagen alignment technique is cheap, fast, precise, and easy to use in comparison to other current techniques. It can be used to align collagen on any type of substrate, such as a gel, which makes it a useful tool in many applications. This technique may also be used to align other extra-cellular matrix proteins and could even be used to create a three dimensional construct with varying fiber orientations.