The quantification of donor-derived cell-free DNA (ddcfDNA) in recipient's plasma is a novel, but technically challenging non-invasive method to assist the diagnosis of acute rejection (AR). A quantitative real-time PCR (qPCR) approach targeting insertion/deletion polymorphisms (INDEL) was adapted to measure ddcfNA in plasma samples from 29 kidney transplant recipients obtained at time of clinically indicated biopsies (8 patients with a histologically verified AR, 9 with borderline rejection and 12 without evidence of rejection). Measured ddcfDNA levels of smaller INDEL amplicon targets differed significantly (p=0.016, Kruskall-Wallis H-test) between recipients with biopsy-proven AR (median 5.24%; range 1.00-9.03), patients without (1.50%; 0.41-6.50) and patients with borderline AR (1.91%; 0.58-5.38). Similarly, pairwise testing by Mann-Whitney U-tests revealed significant differences between recipients with AR and without AR (p=0.012) as well as patients with AR and borderline histology (p=0.015). Receiver operating characteristic (ROC) analysis revealed an area under the ROC curve for discriminating AR and non-AR biopsies of 0.84 (95%CI: 0.66-1.00). The determined cut-off value of 2.7% ddcfDNA showed a sensitivity of 0.88 (95%CI: 0.63-1.00) and specificity of 0.81 (95%CI: 0.64-0.98). INDEL qPCR represents a novel method to quantify ddcfDNA on standard qPCR instruments within 6-8 hours with high sensitivity and specificity to detect AR.