The heparosan polysaccharide serves as the starting carbon backbone for the chemoenzymatic synthesis of heparin, a widely used clinical anticoagulant drug. The previous quantification methods for heparosan rely on time-consuming purification or expensive instruments not readily available for many labs. Here, a chemoenzymatic approach is developed to monitor the production of heparosan in rich medium without purification. After removing the interfering small molecules by ultrafiltration, heparosan was decomposed into oligosaccharides using heparin lyase III. The oligosaccharides were separated from large molecules by ultrafiltration and quantitatively determined by the anthrone-sulfuric acid assay using a spectrophotometer. Based on the different substrate specificity of heparin lyases, the study showed that the concentration of heparosan and heparin in a mixture was discriminatively determined by the two-step chemoenzymatic assay. Furthermore, the anthrone-sulfuric acid assay was observed to be more reliable than the phenol-sulfuric acid assay under these conditions. Besides heparosan and heparin, the chemoenzymatic assay may be adapted to quantify other types of polysaccharides if the specific lyases were available.