Cereal-based bromadiolone anticoagulant is often used for rodent control, and because these baits are attractive for poultry they may be accidentally ingested. Thus, the aim of this study was to develop a new high-performance liquid chromatography (HPLC) method for the determination of bromadiolone residues in hens’ eggs and its plasma kinetics. Laying hens (n = 48) were divided into four groups of 12 animals each. Groups I and II received orally a single dose of bromadiolone 10 mg/kg, group III received a single dose of bromadiolone 60 mg/kg, and group IV was the control. Eggs were collected from groups I, III, and IV, whereas plasma was collected from groups II and IV. The HPLC method developed was reproducible, sensitive, accurate, and linear within the range 0.1–20 μg/g. The final HPLC conditions were as follows: mobile phase MeOH–ammonium acetate (0.5 M) triethylamine buffer (pH 5, 51:49, v/v); analytical column Luna C18 ODS2; wavelength 260 nm; flow rate of 1.5 mL/min; and warfarin as internal standard (5 μg/mL). Recoveries for bromadiolone were in the range of 72–80% with RSD lower than 10%. Pharmacokinetic behavior of bromadiolone in hens results faster than that reported in other animals and humans. Following 10 and 60 mg/kg treatment bromadiolone was not detected in albumen but was present in yolk from day 4 to 5 and from day 2 to 9. In conclusion, the bromadiolone amount found in eggs was well below the toxic dose of this anticoagulant for humans, and no anticoagulant effect should be observed.