As part of the validation of the AmpFlSTR Profiler Plus STR system under reduced polymerase chain reaction (PCR) volume conditions, 275 casework samples were processed. As a means of improving the odds of obtaining balanced and complete profiles for casework samples that show partial profiles or profiles with a slope, several amplification conditions were involved. In addition, a series of 10 simulated mixtures was prepared to establish the limit of detection of a minor profile using the Profiler Plus multiplex system. Two amounts of total DNA (1 and 2 ng) and different ratios that ranged from 1:20 to 20:1 were selected to cover a wide range of potential casework mixtures. The specificity of the Profiler Plus multiplex amplification reaction was also examined under reduced PCR volume conditions by using a large spectrum of nonhuman DNA's, varying the amounts of template DNA (2.5 and 50 ng). All the results indicate the robustness and reliability of the AmpFlSTR Profiler Plus system when used under the experimental conditions described in this report. Reducing the PCR volume to 25 ml allows major cost saving per sample analysis while maintaining high-quality profiles. Thus, very high discrimination can be achieved with minimal analytical consumption of crucial evidentiary samples. 8 figures and 35 references