Background: Up-and-down procedure (UDP) was recommended to replace traditional acute toxicity methods. However, it was limited due to the long experimental period (20–42 days). To improve UDP, an improved UDP method (iUDP) was developed by shortening observation time between sequence dosages. The aim of this study was to test the reliability of iUDP to provide a reliable method for the acute toxicity measurement of valuable or minor amount compounds. Methods: Oral median lethal dose (LD) of nicotine, sinomenine hydrochloride and berberine hydrochloride were measured both by iUDP and modified Karber method (mKM). Results: LD of the three alkaloids measured by iUDP with 23 mice were 32.71 ± 7.46, 453.54 ± 104.59, 2954.93 ± 794.88 mg/kg, respectively. LD of the three alkaloids measured by mKM with 240 mice were 22.99 ± 3.01, 456.56 ± 53.38, 2825.53 ± 1212.92 mg/kg, respectively. The average time consumed by the two methods were 22 days and 14 days respectively. Total grams of the alkaloids used by the two methods were 0.0082 and 0.0673 (nicotine), 0.114 and 1.24 (sinomenine hydrochloride), 1.9 and 12.7 (berberine hydrochloride). Conclusion: iUDP could replace mKM to detect acute toxicity of substances with comparable and reliable result. And it is suitable for valuable or minor amount substances.