The Aspartyl protease of HIV-1 offers an excellent target for the development of specific drugs against the virus. Drugs against protease and reverse transcriptase form the basis for Highly Active Anti-Retroviral Therapy (HAART) that has been successful in improving survival rates and quality of life for HIV infected individuals. However, resistance development to these drugs is a continuing problem, demanding development of additional drugs and approaches to fight virus infection. A thorough understanding of the molecular basis for substrate and inhibitor specificity is critical to defining mechanisms of evasion by drug-resistant mutants as well as for the rational design of drugs able to inhibit a broad spectrum of HIV-1 variants. In this article, we describe characteristics of the protease structure and what is known regarding substrate diversity and mechanisms of cleavage. Approaches to defining substrate diversity are described as an approach to identifying optimal templates for broad-based inhibitor development.