New DNA typing methods need to be thoroughly validated prior to use in forensic investigation. This includes determining the effects different sample conditions have on the typeability of those samples. Biological samples routinely encountered in forensic case work were exposed to a series of different substrates, environmental conditions, and mixtures and typed for the STR HUMTH01 using PCR. None of the conditions resulted in a false typing or preferential allele amplification. It is demonstrated that the application of HUMTH01 typing methods in forensic case work can be reliable, robust, and efficient.