Abstract Misidentification and contamination of biobank samples (e.g. cell lines) have plagued biomedical research. Short tandem repeat (STR) and single-nucleotide polymorphism assays are widely used to authenticate biosamples and detect contamination, but with insufficient sensitivity at 5–10% and 3–5%, respectively. Here, we describe a deep NGS-based method with significantly higher sensitivity (≤1%). It can be used to authenticate human and mouse cell lines, xenografts and organoids. It can also reliably identify and quantify contamination of human cell line samples, contaminated with only small amount of other cell samples; detect and quantify species-specific components in human–mouse mixed samples (e.g. xenografts) with 0.1% sensitivity; detect mycoplasma contamination; and infer population structure and gender of human samples. By adopting DNA barcoding technology, we are able to profile 100–200 samples in a single run at per-sample cost comparable to conventional STR assays, providing a truly high-throughput and low-cost assay for building and maintaining high-quality biobanks.