High-performance liquid chromatography (HPLC) with fluorescence detection was used for the quantification of ivermectin residues in bovine milk intended for human consumption. After liquid–liquid extraction of ivermectin and purification of the extract, the compound was derivatized with 1-methylimidazol in N,N-dimethyl formamide to form a fluorescent derivative, which was separated by HPLC, using reversed-phase C₁₈, with methanol : water (96 : 4 v/v) mobile phase at a flow rate 0.7 ml min⁻¹. The excitation and emission wavelengths of the fluorescence detector were adjusted at 360 and 470 nm, respectively. The linearity of the method was in the range 10–100 ng ivermectin ml⁻¹. Based on a sample of 5.0 ml, the limit of detection and the limit of quantification for ivermectin in milk were 0.6 and 2 ng ml⁻¹, respectively. The recovery rate varied from 76.4 to 87.2%, with an average of 77.9 ± 3.2%, at four fortification levels. The inter-day precision of the method was 13% (n = 5). Of 168 samples analysed, 17.8% contained ivermectin above the limit of quantification. Nevertheless, none of the samples contained ivermectin above the maximum residue limit (10 ng ml⁻¹) established by the Brazilian Ministry of Agriculture.