Titer determination is a prerequisite for the study of viruses. However, the current available methods are tedious and time-consuming. To improve the efficiency of titer determination, we have developed a rapid and simple method for the routine detection of baculovirus titers using a quantitative real-time PCR. This method is based on the amplification of approximately 150-bp fragments located in the coding regions of selected genes. The PCR was found to be quantitative in a range of 103 to 109 virus particles per 200 muL of supernatant, and the results were closely correlated with titers detected from 50% tissue culture infectious doses (TCID50) of baculovirus. This quantitative real-time PCR requires only 30 min to perform, and the entire titer determination can be accomplished within 1 h without the need for cell seeding or further virus dilution and infection. Because this technology is easy to operate, generates data with high precision, and most importantly is very quick, it will certainly be broadly applied for titer determination of baculoviruses in the future.