The determination and application of Analytical Threshold (AT) is a vital part of the forensic Deoxyribonucleic Acid (DNA) internal validation process. AT is the relative fluorescence unit (RFU) signal at which allelic peaks can be confidently distinguished from baseline noise. Several methods of calculating AT are currently being implemented within the forensic DNA community. These methods may utilize DNA negative sample data, DNA positive sample data, or both in their calculations. In this study, two of the DNA positive-based AT calculation techniques were chosen for assessment and comparison. The simplified upper limit approach (ULA) and the cost-based approach. ATs were calculated for each dye channel using a dilution series of 3 single source DNA samples ranging from 0.05-0.8ng. The ATs calculated via the cost-based approach consistently exhibited lower values than those determined via the ULA. As a result, the incidence of allelic drop-out exhibited by these AT values was also consistently lower, with an equivalent or only marginally increased incidence of baseline noise drop-in. These results indicated that the cost-based approach may be a more effective and practical method of calculating AT than the ULA, particularly in the analysis of low DNA template samples.