The purpose of this investigation was to determine milk concentrations of 3 flavonoids found in Scutellaria baicalensis, and to determine whether supplementation of an extract of this plant changes concentrations of these flavonoids in milk. Multiparous Holstein cows (n = 122) were used in a randomized block design to determine responses to dietary S. baicalensis extract (SBE). Treatments were control or administration of SBE (100 g/d of test material). Milk samples were collected on d 3 and once during d 5–12 of lactation. A subset of randomly selected milk samples (5 control and 10 SBE) were used to determine concentrations of baicalin, baicalein, and wogonin. Serial dilutions of standards (100, 50, 25, 10, 5, 2, and 1 µg/mL) were prepared in methanol to generate standard curves. Skim milk samples were subjected to methanol extraction and analyzed by reverse-phase HPLC. Injections of baicalin, baicalein, and wogonin standards showed retention times of 15.4, 21.7, and 28.5 min, respectively. Flavonoids were not reliably detectable below 2 µg/mL, and none of the flavonoids were found at this concentration in milk. The maximum observed concentrations for baicalin, baicalein, and wogonin were 1.69, 0.19, and 0.76 µg/mL, respectively, and only a single sample generated a baicalein peak. Baicalin, baicalein, and wogonin concentrations were adjusted by their respective recoveries (83.3, 48.4, and 27.7% in skim milk) resulting in maximum estimated concentrations of 2.03, 0.39, and 2.74 µg/mL, respectively. Although concentrations were below reliable detection limits, peak area data were analyzed with repeated measures in a mixed model. Peak areas for baicalin and wogonin did not differ by treatment (P = 0.82 and 0.91, respectively) or day of lactation (both P > 0.15). In conclusion, the targeted flavonoids were not readily detectable in milk by this HPLC method, and dietary supplementation of 100 g/d of test material containing SBE showed no evidence of increasing flavonoid concentrations in milk.