The use of biodegradable polymeric nanoparticles (NPs) for controlled drug delivery has shown significant therapeutic potential. Concurrently, targeted delivery technologies are becoming increasingly important as a scientific area of investigation. In cancer, targeted polymeric NPs can be used to deliver chemotherapies to tumor cells with greater efficacy and reduced cytotoxicity on peripheral healthy tissues. In this chapter, we describe the methods of (1) preparation and characterization of drug-encapsulated polymeric NPs formulated with biocompatible and biodegradable poly(D,L-lactic-co -glycolic acid)-poly(ethylene glycol) (PLGA-b -PEG) copolymers; (2) surface functionalization of the polymeric NPs with the A10 2'-fluoropyrimidine ribonucleic acid (RNA) aptamers that recognize the prostate-specific membrane antigen (PSMA) on prostate cancer cells; and (3) evaluation of the binding properties of these targeted polymeric NPs to PSMA-expressing prostate cancer cells in vitro and in vivo. These methods may contribute to the development of other useful polymeric NPs to deliver a spectrum of chemotherapeutic, diagnostic, and imaging agents for various applications.