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% methanolCH2Cl2 and filtrated through 0.22 μm filter. The filtrate was purified through GPC (Gel permeation chromatography). The GPCpurified samples were quantified with HPLC by means of external standard curves. The cytokinins detected in plant tissues were confirmed with LCMS. 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.