Isodesign: A software for optimizing the design of 13-metabolic flux analysis experiments. The growing demand for 13C-metabolic flux analysis (13C-MFA) in the field of metabolic engineering and systems biology is driving the need to rationalize expensive and time-consuming 13C-labeling experiments. Experimental design is a key step in improving both the number of fluxes that can be calculated from a set of isotopic data and the precision of flux values. We present IsoDesign, a software that enables these parameters to be maximized by optimizing the isotopic composition of the label input. It can be applied to 13C-MFA investigations using a broad panel of analytical tools (MS, MS/MS, 1H NMR, 13C NMR, etc.) individually or in combination. It includes a visualization module to intuitively select the optimal label input depending on the biological question to be addressed. Applications of IsoDesign are described, with an example of the entire 13C-MFA workflow from the experimental design to the flux map including important practical considerations. IsoDesign makes the experimental design of 13C-MFA experiments more accessible to a wider biological community. IsoDesign is distributed under an open source license at Biotechnol. Bioeng. 2014;111: 202–208. © 2013 Wiley Periodicals, Inc