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Abstract Background The improvement of multiple myeloma (MM) therapy effectiveness has increased the need for precise, sensitive, and dynamic monitoring of minimal residual disease (MRD). Current MRD assessment methods that are based on bone marrow aspirates, pose limitations due to the invasiveness of the procedure and potential heterogeneous sampling. Recognizing the need for non-invasive, frequent testing, a targeted mass spectrometry, M-InSight®, (MS)-based MRD blood-test was developed. This innovative assay identifies and tracks clonotypic peptides from the patient's monoclonal immunoglobulin, offering a blood-based alternative with equivalent sensitivity to existing bone marrow reliant techniques.Here we employed M-InSight® to sequence and select clonotypic peptides, enabling highly specific, ultra-sensitive quantification of the M-protein. This study examines the correlation between M-protein quantification using M-InSight® and conventional serum protein electrophoresis (SPEP) values routinely obtained in clinical settings. Given the established role of M-protein as a biomarker for MM diagnosis and longitudinal monitoring, ensuring concordance between emerging methodologies and existing standards is vital. Methods Serum samples were obtained from the clinical laboratory at the University of Miami and characterized using SPEP (capillary electrophoresis, Sebia), immunotyping (Sebia), and free light chain quantitation (The binding site). Patients with M-protein concentrations ≥0.2 g/dL by SPEP were included. Longitudinal samples were collected. Subsequently, all samples were analyzed using M-InSight®. The initial sample for each patient underwent de novo sequencing of the M-protein by mass spectrometry and was also used to calibrate the concentration with the known value from SPEP. An in-house bioinformatics algorithm aligned, sequenced, and selected patient-specific clonotypic peptides. These peptides were targeted for the M-inSight® follow up assay allowing the quantification of M-protein. Results A variety of M-protein isotypes were successfully sequenced (IgGK, IgGL, IgAK, IgAL, IgMK, IgML, free K, free L, IgDL, biclonal) at concentrations ranging from 0.2 to 2.9 g/dL. M-protein was successfully sequenced and clonotypic peptides were identified in 57 out of 59 patients using the initial sample. A total of 109 serum samples collected during maintenance or post treatment were analyzed, 50 of them were follow-up samples and were used to correlate M-protein values from both techniques. M-protein quantification by M-Insight showed high concordance with the one obtained by SPEP, with a correlation value of 0.88 (intercept set to 0, slope 0.91. Pearson = 0.886, p-value <0.001). Out of 57 patients whose M-protein was sequenced, 55 were positive by SPEP and were successfully quantified by M-InSight®. Furthermore, M-InSight® detected and quantified M-protein on samples of the 2 remaining patients that showed M-protein values below SPEP detection limits, highlighting the higher sensitivity of M-InSight®. Conclusions M-inSight® assay successfully identified clonotypic peptides in 57 out of 59 patients which were used to quantify M-protein concentration and showed a strong concordance with SPEP and affirming the utility of M-InSight® as a reliable method for routine monitoring of MM. The ability to seamlessly integrate M-InSight® into routine monitoring protocols offers clinicians a valuable tool for MRD monitoring, tracking disease progression and response to therapy with enhanced sensitivity and specificity.