Aims: To validate a real-time PCR test for the diagnosis of Glasser's disease, a major pig disease caused by Haemophilus parasuis. Methods and Results: The specificity of a real-time PCR amplifying the inf B gene was validated with 68 H. parasuis isolates and 36 strains of closely related species. As well, 239 samples of DNA from tissues and fluids of 16 experimentally challenged animals were tested with the real-time PCR, and the results were compared with culture and a conventional PCR. The real-time PCR produced significantly more positive results than the conventional PCR (165 vs 86). Conclusions: The sensitivity of the real-time PCR combined with high specificity makes it a very valuable tool for the diagnosis of Glasser's disease. Significance and Impact of Study: This new method will improve the ability of laboratories to diagnose Glasser's disease, especially in laboratories where the culture method for H. parasuis is not optimal.