The aim of this study was to evaluate the analytical performance of a novel NGS assay, intended for monitoring of donor-derived cell-free DNA (dd-cfDNA), and describe its validity in clinical plasma samples from kidney transplanted patients. Artificial and clinical samples with increasing amounts of patient DNA were evaluated using NGS analysis of indel markers. Monitoring of dd-cfDNA with the NGS assay presented herein demonstrated a sensitivity of ≥0.1% dd-cfDNA and excellent accuracy (R2 0.99) throughout an extensive range of dd-cfDNA (0.1–30%). The precision of the test was determined for two levels (0.1% (LoD) and 1%) of dd-cfDNA. The between run precision (CV%) for the respective level was 16% and 9% and the corresponding result for the within run precision was 19% and 7%. To evaluate performance of the assay in clinical samples, 507 retrospective monitoring samples from 21 patients transplanted either with kidneys from living or deceased donors were analyzed. Monitoring samples were sampled at multiple time points from 24 h up to 90 days post-transplantation. We show that in one patient, increase of dd-cfDNA preceded increase of creatinine caused by acute cellular rejection by several days. In conclusion, the NGS assay displayed a combination of high sensitivity with good accuracy and precision in both artificial and clinical dd-cfDNA samples.