In the field of molecular imaging, selectivity for target cells is a key determinant of the degree of imaging contrast. Previously, we developed a pre-targeted method by which target cells could be selectively imaged using a labeled N-glycan that was ligated in situ with an integrin-targeted cyclic RGD peptide on the cell surface. Here we demonstrate the power of our method in discriminating various cancerous and non-cancerous cells that cannot be distinguished using conventional RGD ligands. Using four cyclic RGDyK peptides with various linker lengths with five N-glycans, we identify optimal combinations to discriminate six types of αβ integrin-expressing cells on 96-well plates. The optimal combinations of RGD and N-glycan ligands for the target cells are fingerprinted on the plates, and then used to selectively image tumors in xenografted mouse models. Using this method, various N-glycan molecules, even those with millimolar affinities for their cognate lectins, could be used for selective cancer cell differentiation.