The sequence-specific retention calculator algorithm (SSRCalc) [ Krokhin , O. V. Anal. Chem. 2006 , 78 , 7785 ] was adapted for the prediction of retention times of N-glycopeptides separated by reversed-phase high performance liquid chromatography (RPLC). The retention time shifts (dHI = HIglyco - HIdeglyco, where HI is the hydrophobicity index, measured in percent acetonitrile units) used for modeling were measured for 602 glycopeptides versus 123 of their deglycosylated analogues. Our method used a tryptic digest of 12 purified glycoproteins, glycopeptide enrichment, deglycosylation with PNGaseF, and RPLC-MS/MS analysis of combined (deglycosylated and intact) peptide mixtures. On average, glycosylation yields a 0.79% acetonitrile unit decrease in retention, compared with the hydrophobicity indices of their deglycosylated analogues. These values, however, are drastically different for asialo (-1.37% acetonitrile units), monosialylated (-0.47% acetonitrile units), disialylated (+0.61% acetonitrile units), and trisialylated (+1.94% acetonitrile units) glycans. Peptide retention time shifts upon glycosylation (dHI) vary depending on the number of monosaccharide units, the presence or absence of sialic acid, peptide hydrophobicity, and the number of position-dependent features. These features are mostly driven by competing effects of acidic residues (aspartic acid and sialic acid) on ion-pair formation and by nearest-neighbor effects of hydrophilic glycans. The accuracy of the modified prediction model for glycopeptides approaches that of the prediction for nonmodified species (R2 = 0.97 vs 0.98). However, retention time prediction based on the experimental retention values of deglycosylated analogues (HIglyco = HIdeglyco + dHI, R2 = 0.995) is much more accurate, thus providing a solid support for glycopeptide identification in complex samples based on mass and retention time.