Spray application is a commonly used, time- and labor-efficient means to deliver live Mycoplasma gallisepticum (MG) vaccine to laying hens in commercial production facilities. The dosage of vaccine received by spray-vaccinated birds can vary due to variation in the spray plume and the vaccine suspension droplet trajectory. In this study, a total of 48 Hy-Line W-36 males were placed individually in isolation units following eye-drop application of gradient levels (1×, 10⁻¹×, 10⁻²×, 10⁻³×, 10⁻⁴×, 10⁻⁵×, 10⁻⁶×, and unvaccinated control) of the MG vaccine. The determined titer associated with a 1× dose was 2 × 10⁶ colony-forming units/dose. Serologic response was assessed weekly following vaccination via serum plate agglutination (SPA) for weeks one through seven postvaccination (p.v.). In addition, immunologic response was assessed at 5, 6, and 7 wk p.v. via MG enzyme-linked immunosorbent assay (ELISA). As indicated by SPA analyses, a 1× dose of vaccine resulted in 100% seroconversion, and dose levels of 10⁻¹× and 10⁻²× resulted in 75% and 37.5% seroconversion, respectively, at 6 wk p.v. The MG ELISA results at 6 wk p.v. demonstrated immunologic responses in 100%, 57.1%, and 28.6% of the 1×, 10⁻¹×, and 10⁻²× dosed birds, respectively. The lower dosage levels of 10⁻³×, 10⁻⁴×, 10⁻⁵×, and 10⁻⁶× did not elicit a response from any bird at 6 wk p.v. Utilizing the SPA data, a logistic regression model was used to determine the relationship between dosage level and seroconversion rate (R²  =  0.999 with a standard error of prediction of 1.6%). The model predicted a required effective dosage of 0.26× for 90% seroconversion at 6 wk p.v. under test conditions.