HIV-1 is an etiological agent of AIDS. One of the targets of the current anti-HIV-1 combination chemotherapy, called highly active antiretroviral therapy (HAART), is HIV-1 protease (PR), which is responsible for the processing of viral structural proteins and, therefore, essential for virus replication. Here, we describe an in vitro transcription/translation-based method of phenotyping HIV-1 PR. In this system, both substrate and PR for the assay can be prepared by in vitro transcription/translation. Protease activity is estimated by the cleavage of a substrate, as measured by enzyme-linked immunosorbent assay (ELISA). This assay is safe, rapid, and requires no special facility to be carried out. Our rapid phenotyping method of HIV-1 PR may help evaluate drug resistance, useful when choosing an appropriate therapeutic regiment, and could potentially facilitate the discovery of new drugs effective against HIV-1 PR.