Protein sialylation is ubiquitous and essential in a wide range of biological processes. Herein, a mass defect-based chemical-directed proteomics method (MdCDPM) was presented for targeted analysis of intact sialylglycopeptides (SGPs). The process starts by specific oxidation of dihydroxy in sialic acid to aldehyde, which was then chemically labeled by two arginine isotopologues (Arg-15N4 and Arg-D4, differs by 36 mDa). The equally mixed precursor partners, spacing tens of mDa apart, enable the direct recognition of SGPs in MS1 level and benefit the subsequent targeted MS2 characterization. The mass envelope of two labeled forms falling into a narrow m/z window strengthens recognition uniqueness greatly, and the proposed 1:1 intensity ratio of doublets will not be readily distorted. More important, such subtle mass differences permit multiple sialic acids labeling without additional complexity of precursor patterns. Also, the partner m/z shifts detail the number of sialic acids contained in the precursor species. By applying MdCDPM, femtomole quantities of SGPs could be detected from total cell lysates, even at a signal-to-noise ratio of as low as 3:1. In addition, assays were performed to estimate the false positive rate and demonstrated high confidence of MdCDPM. Furthermore, it was designed and successfully exploited to analyze SGPs in human serum, which highlighted the feasibility of this strategy for biological applications.