Periodontal disease is one of the most frequently encountered chronic inflammatory disease of the oral cavity. Current research data imply that the pathogenesis of periodontal disease is mediated by the host response giving the rationale for the usage of locally applied corticosteroids, such as betamethasone dipropionate (BDP), in periodontal therapy. Gingival crevicular fluid (GCF), an inflammatory exudate formed during the course of periodontal disease, is a valuable matrix for therapeutic monitoring of chemotherapeutic agents used in periodontal therapy. In this book, the development, optimization and validation of a simple RP-HPLC method for determination of BDP in GCF is described. The proposed method has been successfully applied for determination of BDP concentration in GCF samples obtained from patients with chronic periodontal disease after the local treatment with BDP cream 0.5 mg g-1. This book may serve as guide for development and optimization of analytical methods which may be used for determination and monitoring of other molecules in GCF samples, thus presents a contribution in rational therapy of periodontal disease.