Metabolic flux analysis (MFA) represents a powerful tool for systems biology research on eukaryotic cells. This review describes recent advances, the challenges as well as applications of metabolic flux analysis comprising fungi, mammalian cells and plants. While MFA is widely established and applied in microorganisms, it remains still a challenge to adapt these methods to eukaryotic cell systems having a higher complexity particularly concerning compartmentation or media composition. In fungi MFA was used in the past few years to analyze a variety of conditions and factors and their effects on cellular metabolism. In mammalian cells MFA was applied mainly in cell culture technology and in medical and toxicological research. 13C metabolic studies on native whole plants are additionally challenging by the fact that CO2 is usually the only carbon source.