Abstract: Method of simultaneous determination of ephedrine and calchicine in blood and urine by HPLC-MS in hyphenation was proposed.Ephedrine and colchicine in blood or urine sample were extracted with a mixture of trifluoroacetic acid and acetonitrile (1+99),purified the Waters Oasis MCX micro-column and separated on the XDB-C8 column,using a mixture of methanol and 0.04% trifluoroacetic acid solution (33+67) as mobile phase.Determination was made by using ESI and under the mode of SIM.The ions of quantitative analysis were:ephedrine (m/z) 166M+H+,calchicine (m/z) 400M+H+.Linearity were held in the ranges of 4.0-1 000.0 μg·L-1 (for ephedrine) and of 6.0-1 000.0 μg·L-1 (for calchicine),with detection limits of 4.0 μg·L-1 and 6.0 μg·L-1 respectively.Recoveries obtained were in the range of 81.0%-109.0% for both compounds.The intra-day RSD and inter-day RSD found were <6.4% and <9.9% respectively.