Carbohydrates are the most abundant class of biomolecules on earth, performing numerous biological essential roles (e.g., energy source, energy storage, and structural components). The recent application of advanced analytical techniques, especially high-performance anion-exchange chromatography coupled with a pulsed amperometric detector (HPAEC-PAD), to analyze carbohydrates has enabled researchers to characterize and quantify carbohydrates in different matrices (e.g., foods/beverages, products from bioprocesses, and body fluids). HPAEC-PAD is a versatile, relatively simple, fast, sensitive, and selective technique to analyze and characterize different carbohydrates. We describe herein HPAEC-PAD methods that have been used successfully to investigate the profile and content of the most common carbohydrates. Specifically, we used food matrices as a model for carbohydrate analysis by HPAEC-PAD. We aim to provide the reader with guidelines on applying the HPAEC-PAD technique to identify and quantify different carbohydrates and highlight this approach’s advantages and limitations.