Routine protein assays are usually affected with various compounds, and we need to use different protein quantification protocol to deal with different interference. In order to simplify the procedure, we developed a new method, in which the components and concentrations of the reagents were modified mainly based on classic Folin-Ciocalteu’s reagent for reducing the susceptibility to interfering substances. Standard curves of the new method were established with different levels of bovine serum albumin, and then, we assessed and evaluated the detectable wavelengths and stability. In particular, the tolerability to several interfering substances was analyzed by using cytolysis solutions containing different chemicals. Our data in this study show that the new method could be applied to detecting protein concentrations accurately, even in the presence of surfactants such as 10% sodium dodecyl sulfate (SDS), 2% NP-40, or 1% TrintonX-100, chelators of 25 mmol/L EDTA or 1 mmol/L Ethylene glycol bis (2-aminoethyl) tetraacetic acid (EGTA), reductants of 1 mmol/L Dithiothretol (DTT) orβ-Mercaptoethanol (ME), or nitrogen-containing compounds of 0.5 mol/L ammonium sulphate or 4?mol/L urea. Taken together, these results indicate that the new approach significantly improves the tolerance to the interfering substances, which could be potentially useful in measuring the contents of proteins interfered with such substances.