Valerian root (Valeriana officinalis) is an herbal tranquilizer used in horses. However, many plants and herbs are classified as forbidden by the USEF Equine Drugs and Medications Rule due to their potential to affect performance. The aim of this study was to establish a reliable withdrawal period before competition for trainers considering valerenic acid supplements as a training aid. Ten mares between 10 and 20 years of age were administered an oral paste of valerian root extract (36 mg) daily for 5 consecutive days. Urine samples were collected by sterile catheterization before the first and all subsequent treatments. Urine was also collected at 4, 8, 24, 48, and 72 h after the last treatment. Urine samples were coded for blinding and stored at 4°C until testing. Valerenic acid in urine was analyzed using a triple-quadrupole mass spectrometer coupled to an Agilent 1100 capillary HPLC system. Quantitation of urine valerenic acid was done by generating a calibration curve using the peak areas of 3 replicate injections for each calibration point. Two different calibration curves for quantitation of valerenic acid in urine were created. One curve was generated by spiking valerenic acid at 0 (blank), 25, 50, 100, 200, and 400 ng/mL into known blank urine before protein precipitation and solid-phase extraction. A second curve was generated for later samples by pooling 2 mL from each of 4 blank urine samples and then adding valerenic acid as described. Regression analysis of a representative calibration curve generated from these standards yielded a linearity of R² = 0.9969. Based on the signal-to-noise ratio of the 25 ng/mL calibration standard, the limit of quantitation was estimated at 20 ng/mL. From a total of 90 post-valerenic acid treatment urine samples, 3 samples were positive. Urine samples obtained from one horse was positive for valerenic acid on d 2 (21 ng/mL) and 3 (29.5 ng/mL) of treatment. A different mare tested positive (67.8 ng/mL) 4 h after the last dosage (d 5). Based on our results, horses should be withdrawn from competition for at least 5 d after the oral administration of 36 mg valerenic root extract.