We have previously reported a novel method for the selective detection of antibody Fab, named nano-surface and molecular-orientation limited (nSMOL) proteolysis. The chemistry of nSMOL is Fab-selective limited proteolysis by making use of the difference of the protease nanoparticle diameter (200 nm) and antibody resin pore diameter (100 nm). In this report, we have demonstrated the full validation of trastuzumab bioanalysis in human plasma using an nSMOL protocol coupled with LC-MS/MS. The immunoglobulin fraction was collected by using Protein A resin, then the nSMOL reaction was performed using FG nanoparticle-immobilized trypsin under nondenaturing physiological conditions at 50 °C for 7 hours. After the removal of resin and nanoparticles, the tryptic peptides were separated with a C18 column (2.1 × 50 mm, 1.9 μm, and 20 nm pore) using the mobile phase of 0.1% aqueous formic acid and acetonitrile. The signature peptides of the trastuzumab complementarity-determining region (CDR) and P₁₄R internal standard were simultaneously quantified by LCMS multiple reaction monitoring. This nSMOL method showed that the lower limit of quantification (LLOQ) of trastuzumab was 0.977 μg ml⁻¹ and the linear dynamic range was from 0.977 to 250 μg ml⁻¹ in plasma. The intra- and inter-assay precision of the LLOQ, low quality control (LQC), middle quality control (MQC), and high quality control (HQC) was 2.2–13.1% and 10.2%, 3.8–7.6% and 8.2%, 2.6–6.7% and 4.4%, and 4.9–5.7% and 5.6%, respectively. The nSMOL approach with LC-MS/MS has the potential to become the standardized method for the bioanalysis of any monoclonal antibody.