Intracellular metabolic fluxes are important to understand metabolic characteristics of cells and to direct metabolic engineering strategies. Because intracellular fluxes cannot be directly assessed, isotope experiments are usually conducted to trace metabolic fluxes. The flux-ratio analysis can reflect high biochemical veracity, be employed to identify the topology of the networks, and offer greatly reduced computational expense for flux determination. In order to apply this metabolic analysis method to better elucidate more cell systems, we discussed in this study the principles, experiments and assays, data interpretation, and other issues that should be considered in flux ratio determination, metabolic flux quantification and its metabolic engineering applications.