An interlaboratory study of 21 public health, state agriculture, and industry laboratories in the United States tested raw commingled bovine milk containing aflatoxin M1 using the Charm Rapid One Step Assay (ROSA) Safe Level Aflatoxin M1 Quantitative lateral flow method. Blind coded sample pairs were fortified with 0, 300, 350, 400, 450, 500, and 550 parts per trillion (ppt) aflatoxin M1. A ROSA reader quantitatively interpreted test strips with ppt readings. Readings < or = 400 ppt were interpreted as negative, and readings >400 ppt were interpreted as positive. Initial positive samples were subsequently assayed 2 additional times. If both retest results were >400 ppt, the sample was called positive/ actionable relative to U.S. and Codex levels, 500 ppt. The concentration of 400 ppt was chosen for the positive/negative interpretation to provide 90% sensitivity with 95% confidence at the 500 ppt legislative level. The combined false negative rate was <5% (4 of 83) for samples at 500 and 550 ppt. The false violatives at 0, 300, 350, 400, and 450 ppt (n = 42 at each level) were 0, 0, 21, 14, and 93%, respectively. The 90% positive concentration with 95% confidence was 503 ppt by probit analysis. The average intralaboratory repeatability was 11% and average interlaboratory reproducibility was 13% for the fortified sample pairs. High-performance liquid chromatography analysis of the study samples by 5 laboratories showed 38% false negatives with the 500 and 550 ppt samples, and a 0% false-violative rate with samples less than the 500 ppt action level.