Reliable methods for detection of A. galli infection using excreta egg count (EEC) and ELISA assays to determine A. galli specific IgY levels in serum and yolk samples were compared from hens infected naturally and artificially. Artificially infected hens were used to generate samples for analysis of preferred detection methods and to generate contaminated ranges for use in the naturally acquired infection study in which Lohmann Brown hens (n = 200) at 16 weeks of age were randomly assigned to four treatments with five replicate pens. Hens of negative control (NC) ranged on a decontaminated area, hens of low infection, medium infection and positive control (PC) ranged on the areas previously contaminated by hens artificially infected with 250, 1000 and 2500 A. galli eggs/hen, respectively. Additionally, hens of PC were orally infected with 1000 A. galli eggs/hen. Anti A. galli antibody levels in hen serum (SIgY) and yolk (YIgY) were measured before range access, and 2, 7 and 12 weeks after access to the contaminated ranges. In a natural infection study, eggs were detected in the excreta of all hens 4 weeks after range access, with the exception of NC in which no eggs were detected. EEC increased to reach maximum value (2204 ± 307 eggs/g) after 11 weeks of range access and then declined at 12 weeks (905 ± 307eggs/g) (p < 0.01). While SIgY OD values were not different in hens between any groups before range access, after 2 weeks, both SIgY and YIgY gradually increased in hens of PC (1.17 ± 0.03 and 0.88 ± 0.04) and medium infection (1.07 ± 0.03 and 0.96 ± 0.04) compared to low infection (0.38 ± 0.03 and 0.29 ± 0.04) (p < 0.01) and NC. After 12 weeks, SIgY were similar in hens of PC, medium and low groups whereas YIgY was higher in hens of low infection group (p < 0.01). Sensitivity of the serum and egg yolk antibody levels assay to detect A. galli infection was 100% and 96%, respectively, whereas the pooled EEC method yielded a sensitivity of 93%. The results of this study suggest that hens naturally infected with A. galli produce both SIgY and YIgY at different levels depending on the infection intensity and duration of exposure which allows the diagnosis of prior infection or early diagnosis of current infection. Use of the practical and non-invasive method of yolk sample analysis for detecting IgY can be just as informative as using serum samples to detect A. galli infection.