The sugar cane industry has seen how biomass production in sugar mills would be converted to a readily available source of molecules besides sugar. Properly managed, byproducts would be transformed into a sustainable source of renewable and environmentally friendly chemical products. As a principal and more abundant organic acid in sugar cane juice, trans-aconitic acid (TAA) has been studied for use as a plasticizer in the polymer industry. However, up to now no industrial-scale application has been reported. As a reasonable approach to recover TAA from a sugar mill, first, an analytical method to determine its presence in all stages of the sugar-manufacturing process is needed. A new modern method was developed to measure TAA in seven stages in a sugar mill located in Valle del Cauca, Colombia. The stages with higher content of TAA were syrup, with 3363.6 ± 589.3 mg/L, and honey (molasses), with 6110.05 ± 139.5 mg/L.