Animal models testing the ability of vaccines and therapeutic agents to prevent pathology from induced respiratory infection are an important means of testing and validating the vaccines and therapeutic agents. However, the lack of induced pathology in test subjects could be either indicative of protection or a problem with the animal model system. This work describes the improvement of a chicken model system of intratracheal infection using fluorescent microspheres as a positive indicator of infection. It was shown that fluorescent microspheres and Mycoplasma gallisepticum bacteria both dispersed to the same areas of the chicken respiratory system and that the microspheres remained detectable in the chicken lung tissue for at least 7 days following infection. The microspheres used are detectable using a black light, allowing for visualization during necropsy. Using the updated model system, three live M. gallisepticum vaccines were tested both for their ability to elicit a humoral immune response following vaccination, and for their ability to protect from air sac lesion pathology at two different time points following vaccination. Results showed the protective effects of the different M. gallisepticum vaccines prevented the induction of pathology, consistent with previous results. The presence of the fluorescent microspheres provided a positive method of identifying the properly infected chickens and a means of differentiating failed experimental infections so that those samples could be removed, resulting in improved consistency in infection results.