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ABSTRACT: Cell-free transcription-translation systems (TXTL) are becoming reliable tools to rapidly prototype biochemical systems programmed with synthetic gene circuits. Here we scale down TXTL reactions to 0.5 μL using the Echo® 550 Liquid Handler, and compare the results to 5 μL reactions prepared using manual methods. Using reaction condition optimization and gene circuit prototyping, we demonstrate that data obtained via miniaturization with the Echo 550 system is equivalent or superior to corresponding manual methods at larger volumes. Miniaturization reduces the cost per reaction up to a factor of 10. By implementing the Echo system we also reduce reaction setup time and work load on the individual researcher.