The demonstration of on-site STR analysis showed the feasibility of real-time DNA typing for the identification of probative biological evidence at a crime scene. In order to evaluate the capabilities of the portable microsystem developed and its compatibility with crime-scene investigation processes, real-time STR analyses were conducted at a mock crime scene prepared by the Palm Beach County Sheriff’s Office (Florida). Blood stain sample collection, DNA extraction, and STR analyses with the portable microsystem were conducted in the field, followed by a successful “mock” hit on CODIS (Combined DNA Index System). The DNA match with a suspect was obtained within 6 hours of the sample collection. The portable micro device consists of a 160-nL polymerase chain reaction (PCR) reactor with an on-chip heater and a temperature sensor for thermal cycling, microvalves for fluidic manipulation, a coinjector for sizing standard injection, and a 7-cm-long separation channel for capillary electrophoretic analysis. A nine-plex autonomic STR typing system that consists of amelogenin and eight CODIS core STR loci was constructed and optimized for this real-time human identification study. Reproducible STR profiles of control DNA samples were obtained in 2 hours and 30 minutes with <0.8 bp allele typing accuracy. The minimal amount of DNA required for a complete DNA profile is 100 copies. The detailed descriptions of materials and methods address the microdevice and instrument; PCR amplification and capillary electrophoresis; data acquisition and analysis; DNA extraction, quantification, amplification, and DNA profile verification; mock crime-scene setup and evidence collection; and DNA analysis at the mock crime scene. 6 figures and 40 references