Allelic quantification of STRs, where the presence of three or more alleles represents mixtures, provides a novel method to identify mixtures from unknown biological sources. The allelic stutters resulting in slightly different repeat containing products during fragment amplification can be mistaken for true alleles complicating a simple approach to mixture analysis. An algorithm based on the array of estimated stutters from known samples was developed and tuned to maximize the identification of true nonmixtures through the analysis of three pentanucleotide STIRS. Laboratory simulated scenarios of needle sharing generated 58 mixture and 38 nonmixture samples that were blinded for determining the number of alleles. Through developing and applying an algorithm that additively estimates stuttering around the two highest peaks, mixtures and nonmixtures were characterized with sensitivity of 77.5, 82.7 and 58% while maintaining the high specificity of 100, 97.4 and 100 for the W, X, and Z STIRS individually. When all three STRs were used collectively, the resulting sensitivity and specificity was 91.4 and 97.4%, respectively. The newly validated approach of using multiple STIRS as highly informative biomarkers in unknown sample mixture analyses has potential applications in genetics, forensic science, and epidemiological studies.