Inter-laboratory validation studies were conducted in 5 laboratories to validate the biological method for determination of tetracyclines in royal jelly. Oxytetracycline spiked at the levels of 0.2 and 1.0 ppm was analyzed. Mean recoveries were 88 and 90%, reproducibility relative standard deviations (RSD(R)) were 13.7 and 7.8%, and HORRAT(R) values were 0.7 and 0.5. Samples containing residues at the levels of 0.25 and 0.80 ppm were analyzed. Mean recoveries were 73 and 77%, RSD(R) were 12.6 and 10.5%, and HORRAT(R) values were 0.6 and 0.6. The determination limit was 0.1 ppm (oxytetracycline, tetracycline) and 0.02 ppm (chlortetracycline). These results show that this method has good performance.