Accurate determination of residual protein content in hydroxypropyl chitin (HPCH), a temperature-sensitive chitin derivative developed recently, is of great importance for biomedical applications. Coomassie brilliant blue (Bradford) method, Lowry method and Bicinchoninic acid (BCA) method were investigated to estimate the content of residual protein in HPCH. It was found that both Bradford and Lowry methods are not suitable for the protein assay in the HPCH sample. The BCA method could be used to determine protein content in HPCH solutions at low concentrations with good accuracy and precision and reasonable limit of detection and limit of quantitation for medical applications. Display omitted •Bradford and Lowry methods not suitable for the protein assay in the HPCH sample.•BCA method used to analyze protein in HPCH solutions with good accuracy and precision.•Low CHPCH chosen in BCA assay with reasonable LOD and LOQ for medical applications.