Next generation human therapeutic monoclonal antibodies (t-mAbs) are harder to quantify with the widely used bottom-up tryptic digestion method. Due to their homology with endogenous immunoglobulins, there is a lack of unique and stable ‘signature’ peptides that can be targeted. Middle-up two dimensional liquid chromatography high resolution mass spectrometry (2D-LC-HRMS), targeting the entire light chain, was examined as an alternative. Adalimumab (ADM) was successfully quantified in human plasma after Melon® Gel sample purification, followed by orthogonal separation on a weak cation exchange (WCX) and reversed phase column. Charge and hydrophobicity were used to separate ADM from the polyclonal immunoglobulin background. HRMS with its high resolution and exact mass was able to isotopically resolve the ADM light chain and to provide another separation dimension on the basis of mass to charge ratio. Using the targeted single ion monitoring (T-SIM) with multiplex (MSX) option, three ADM light chain precursors, 2341.80, 2129.00, and 1951.68 m/z, and one internal standard precursor 2146.39 m/z, were measured simultaneously. The Melon® Gel sample purification was found to be very efficient in removing plasma proteins that would otherwise interfere with chromatographic separation and ionization. The linearity of the method for the analysis of ADM was excellent (R2=0.999) between 1 – 128 mg/L with an LLOQ signal to noise ratio (S/N) of 10. Within-run and between-run precision and accuracy were in concordance with the EMA guideline. Cross-validation of the 2D-LC-HRM method with the standard peptide LC-MS/MS method showed a good agreement (R2 = 0.86) between the methods. However, there was a bias present, possibly due to charge variant ADM formation over time. Since the presented 2D-LC-HRMS method is able to measure only the native form of ADM, it is able to provide a measure of the active form of ADM in patients.