The growing interest in G-quadruplex (G4) structure and function is motivating intense efforts to develop G4-binding ligands. This chapter describes the design and testing of peptide nucleic acid (PNA) oligomers, which can bind to G4 DNA or RNA in two distinct ways, leading to formation of heteroduplexes or heteroquadruplexes. Guidelines for designing G4-targeting PNAs and step-by-step protocols for characterizing their binding through biophysical or biochemical methods are provided.