Scratch wound healing assays remain one of the most commonly used 2D migration assay to obtain a broad overview of the migration behavior of cultured cells. They are easy to perform and cost effective, but yet not standardized in terms of the geometric dimensions of the scratch, e.g. consistency of the scratch width and straightness of the line, especially when performed manually. Furthermore, conventional scratch assays only include a single scratch, which leads to a limited evaluation of a restricted cell population within the culture plates, but not of all cells grown in the chamber. There are commercially available ′scratch-makers′ which are highly expensive and yet lack advanced functions such as scratching complex patterns. However, such complex scratch formations would make it possible to assess the entire cell population in a chamber and to evaluate the influence of the scratch itself, e.g. with regard to the regulation of migration-associated proteins. We have therefore developed for the first time a method that not only standardizes the scratch dimensions for different pipette tips, but also gives the opportunity to scratch any conceivable 2D shape by using 3D printers as 2D plotters. This open-source and low-cost system can be performed with any plate format from any manufacture under culture hood conditions. The 2D plotter-based method developed and validated by us provides highly reproducible and consistent scratch parameters for in vitro migration analyses.