The amino acid sequence of the M-protein for multiple myeloma is unique compared to the polyclonal antibodies in patients’ blood. This uniqueness is exploited to develop an ultra-sensitive M-protein detection method utilizing mass spectrometry (MS). The method involves the de novo amino acid sequencing of the full-length M-protein, and a targeted MS/MS assay to detect and quantify the unique M-protein sequence in serum samples. Healthy control serum spiked with NISTmAb, and serial samples from an MM patient are used to demonstrate the ability of the platform to sequence and monitor a target M-protein. The de novo NISTmAb protein sequence obtained matched the published sequence, confirming the ability of the platform to accurately sequence a target M-protein in serum. NISTmAb was quantified down to 0.0002 g/dL in serum, hundreds of times more sensitive than conventional blood-based tests such as SPEP and IFE. The M-protein in the patient sample could be quantified throughout complete remission, demonstrating the utility of the assay to track M-protein considerably beyond the sensitivities of current blood-based tests. Notably, the assay detected molecular relapse 10 months before detection by IFE. The MS-based assay is highly sensitive, non-invasive and requires only a small amount of serum, less than 100 μl, to monitor M-protein levels in MM patients.