To take full advantage of the unique optical properties of quantum dots (QDs) and expedite future near-infrared fluorescence (NIRF) imaging applications, QDs need to be effectively, specifically and reliably directed to a specific organ or disease site after systemic administration. Recently, we reported the use of peptide-conjugated QDs for non-invasive NIRF imaging of tumor vasculature markers in small animal models. In this protocol, we describe the detailed procedure for the preparation of such peptide-conjugated QDs using commercially available PEG-coated QDs and arginine-glycine-aspartic acid (RGD) peptides. Conjugation of the thiolated RGD peptide to the QDs was achieved through a heterobifunctional linker, 4-maleimidobutyric acid N-succinimidyl ester. Competitive cell binding assay, using 125I-echistatin as the radioligand, and live cell staining were carried out to confirm the successful attachment of the RGD peptides to the QD surface before in vivo imaging of tumor-bearing mice. In general, QD conjugation and in vitro validation of the peptide-conjugated QDs can be accomplished within 1–2 d; in vivo imaging will take another 1–2 d depending on the experimental design.