Background. Determining the distributive fate and retention of a cell therapy product after administration is an essential part of characterizing it's biosafety profile. Therefore, regulatory guidelines stipulate that biodistribution assays are a requirement prior to advancing a cell therapy to the clinic. Here the development of a highly sensitive quantitative polymerase chain reaction (qPCR)-based method of tracking the biodistribution and retention of human mesenchymal stromal cells (hMSCs) in mice, rats or rabbits is described. Methods. A primer-probe based qPCR assay was developed to detect and quantify human Alu sequences in a heterogeneous sample of human DNA (hDNA) and murine DNA from whole organ genomic DNA extracts. The assay measures the amount of genomic hDNA by amplifying a 31 base pair sequence of the human Alu (hAlu) repeat sequence, thus enabling the detection of 0.1 human cells in 1.5 x 10(6) heterogeneous cells. Results. Using this assay we investigated the biodistribution of 3 x 10(5) intramuscularly injected hMSCs in Balb/c nude mice. Genomic DNA was extracted from murine organs and hAlu sequences were quantified using qPCR analysis. After 3 months, hDNA ranging from 0.07%-0.58% was detected only at the injection sites and not in the distal tissues of the mice. Discussion. This assay represents a reproducible, sensitive a method of detecting hDNA in rodent and lapine models.This manuscript describes the method employed to generate preclinical biodistribution data that was accepted by regulatory bodies in support of a clinical trial application.