Nanoparticles (NPs) are materials with overall dimensions ranging between 1 and 100 nm. The application of NPs in medicine has been extensively explored in recent years and had a significant impact on treatment of many diseases, especially cancer. NPs are used to overcome the limitations of standard therapies involving free drug molecules, and they are primarily used to shield or solubilize highly toxic or hydrophobic agents, increase their bioavailability, prolong blood circulation, and increase drug delivery to diseased cells. Also, NPs are highly sensitive contrast agents and were applied for diagnostic purposes using noninvasive imaging modalities, such as ultrasound, optical, or magnetic resonance imaging (MRI). Herein, we describe a novel synthesis method to formulate biodegradable polymeric NPs coated with short hexapeptides. This is a pioneering methodology, where the peptides serve a dual function: to stabilize the NP and target cancer cells. The peptide-coated NPs are stable in serum and do not induce oxidative stress in a biological setting. We outline the synthesis protocol for the NPs and demonstrate their applications as optical contrast agents in imaging of cancer cells.