Abstract: A novel method to determine in situ the changes in constituents of 3-bromthiophene, 3-alkylthiophenes (3-butylthiophene, 3-hexylthiophene, 3-octylthiophene， 3-isooctylthiophene， 3-dodecylthiophene) and by-products during the alkylated reactions was developed using a reversed-phase high performance liquid chromatography. After washed with water, extracted and flash distillation, 3-alkyl thiophenes were analyzed by RP-HPLC-photo diode array detector and quantified by external standard method under the critically examined chromatographic conditions. 3-Alkyl thiophene could be completely separated from 3-bromthiophene and other by-products without interference from each other. All the linear ranges of five 3-alkyl thiophenes and 3-bromthiophene were 0.100-10.0 mg/L with the correlation coefficients of 0.9952-0.9993 and the limits of detection of 20.0 g/kg (S/N=3). The average recoveries and relative standard deviations（RSD）were in the ranges from 89.2% to 102.6% and 1.04% to 4.98%, respectively. The results indicated that the proposed method was easy, fast, accurate, and could meet the requirement for determination of 3-alkylthiophenes and be applied for tracing the changes of raw material and products during preparation, which might in situ provide available information for reaction kinetics research.