Abstract: OBJECTIVE To establish a rapid detection method for spore counting of viable organism preparation of Bacillus licheniformis by HPLC. METHODS HPLC was adopted to determine dipicolinic acid(DPA) which was released by Bacillus licheniformis, using a Waters Xbridge C₁₈ column(4.6 mm×250 mm, 5 μm) with mobile phase consisting of 0.1% phosphoric acid-methanol(92:8), while the column temperature was maintained at 30℃, the flow rate was 0.8 mL·min^-1 and the detection wavelength was 273 nm. Plate counting method was adopted to determine spores in corresponding spore suspension in order to establish a relationship curve between the concentration of DPA and spore counting. The methods were applied to detect the spore count in three viable organism preparations of Bacillus licheniformis(tablet, capsule and granule), at the same time. RESULTS Stability, precision, repetition and recovery rates of the method established were all good. The standard curve of DPA was linear over the range of 2.5-80 mg·L^-1(r=0.999), with the average recovery rate of 102.3%(n=9). There was a linear relationship between the concentration of the spore counting and DPA with a range of spore counting concentration of 6.5×10⁸-7.8×10⁹ CFU·mL^-1(r=0.993). There were no significant differences in spore counting of three viable organism preparations of Bacillus licheniformis(tablet, capsule and granule). CONCLUSION Method of rapid detection for spore counting of viable organism preparation of Bacillus licheniformis is well worked, simple and rapid.