Abstract: Using leaves originated from Populus popularis cuttings and Nicotiana tabacum plantlets, the research aimed to develop a GPC-HPLC method to determine the concentration of cytokinin in plant tissues. According to the reclaim efficiency of standard cytokinin, the recommended procedures were as follows: plant samples were grinded with 80% methanol and extracted at 4℃ overnight. Crude extract was filtrated and then centrifugated. The liquid was added with two drops of ammonia, and then vacuum dried at 40℃. The samples were resolved by 3% methanolCH2Cl2 and filtrated through 0.22 μm filter. The filtrate was purified through GPC (Gel permeation chromatography). The GPCpurified samples were quantified with HPLC by means of external standard curves. The cytokinins detected in plant tissues were confirmed with LCMS. The results showed that the contents of zeatin in poplar and tobacco leaves were 439.501 8 and 617.995 7 ng/g FW, respectively. Kinetin (49.473 9-124.712 4 ng/g FW) was found in plant tissues, presumably resulting from the uptake from tissue culture medium of tabacco and insects feeding (aphid) in poplar.