Added PubMed ID Liquid chromatography coupled mass spectrometry (LC-MS) operating in targeted mass spectrometry (MS) including multiple reaction monitoring (MRM) mode is an accurate analytical method. Integrating with nanoelectrospray-tandem MS (NanoES-MS/MS) and immunoprecipitation (IP), it becomes a promising method in detecting serum biomarkers which are at extremely low concentrations, such as mycobacterium tuberculosis (Mtb) secreted 10 kDa culture filtrate antigen (CFP-10). We employed an optimum denaturing condition for trypsin digestion and IP of targeted peptides. The condition shows several advantages over commonly used one, 1) it prevents further dilution of sample, thus enables as-needed sample volume, 2) It ensures liberation of trace amount of antigen peptide under a cost-effective enzyme to substrate mass ratio, 3) It avoids time-consuming and unnecessary reduction of disulfide bonds, as some pathogen antigens don't contain disulfide bond. More importantly, optimum MRM transitions of the targeted peptide and standardized data processing and interpretation algorithm provide confirmative information of it in serum samples. By changing the denaturing buffer, addition of a clean-up step, selection of optimum MRM transitions and better interpreting MS signal, this workflow showed an improved accuracy in clinical samples.