The PrepFiler Forensic DNA Extraction Kit (“the kit”) enables the isolation of DNA from biological samples that contain small quantities of biological material. The kit facilitates reversible binding of DNA with magnetic particles, resulting in high DNA recovery from samples with very low and high quantities of biological material (0.1 and 40 mL of human blood provided 14 and 2883 ng of DNA, respectively). The kit also obtains the DNA at a high concentration, so that the volume of extract used for polymerase chain reaction (PCR) is minimal. In addition, the kit removes PCR inhibitors or substances that interfere with the PCR and can extract DNA from a variety of biological samples. The manual protocol and its chemistry can be adapted to automation. The testing of the kit was done by evaluating the extraction of genomic DNA from various sample types commonly processed in a forensic laboratory. Forensic-type samples were prepared by using various substrates and saliva, blood, and semen obtained from human donors. The forensic-type samples included inhibited samples and samples that had been exposed to adverse chemical and environmental influences. Detailed descriptions of materials and methods address the extraction and quantification of DNA, STR analysis, the extraction of blanks, sensitivity studies, the reproducibility study, stability studies, case-type samples, and correlation studies. 4 tables, 8 figures, and 33 references