Over the past few decades, panels of human cancer cell lines have made a significant contribution to the discovery and development of anticancer drugs. The National Cancer Institute 60 (NCI60), which consists of 60 cell lines from various human cancer types, remains the most powerful human cancer cell line panel for high throughput screening of anticancer drugs. The development of JFCR39, comprising a panel of 39 human cancer cell lines coupled with a drug-activity database, was based on NCI60. Like NCI60, JFCR39 not only provides disease-oriented information but can also predict the action mechanism or molecular target of a given antitumor agent by utilizing the COMPARE algorithm. The molecular targets of ZSTK474 as well as several other antitumor agents have been identified by using JFCR39 and some of these compounds have since entered clinical trials. In this review, we will describe human cancer cell line panels particularly JFCR39 and its application in the discovery and/or development of anticancer drug candidates.