Hydroxychloroquine is a medication used to treat autoimmune conditions. Overdoses of hydroxychloroquine are uncommon, with most recommendations on monitoring drawing from experience with more common overdoses of the related drug chloroquine. We present a case of an adolescent with intentional overdose of approximately 12 g of hydroxychloroquine. The prominent clinical features were hypokalemia and widened QRS and QT intervals on the electrocardiogram. Therapy included epinephrine by intravenous drip and bicarbonate infusions along with supportive care and cardiac monitoring. The patient recovered without sequelae. Urine drug testing showed an absorbance alarm for one of the components of the institution drug of abuse screening panel, an oxycodone screen using an enzyme immunoassay. Analysis of two urine specimens collected during the hospitalization revealed hydroxychloroquine concentrations of greater than 500 mg/L (approximately 7.5 h after ingestion) and 130 mg/L (approximately 14 h after ingestion). Only the urine with greater than 500 mg/L hydroxychloroquine produced absorbance alarms on the drug of abuse testing. We separately analyzed the impact on 24 urine assays of varying concentrations of hydroxychloroquine spiked into de-identified pooled urine samples. For 6 of the assays (buprenorphine, cotinine, oxycodone, and tetrahydrocannabinol qualitative drug screens; microalbumin and urine myoglobin quantitative assays), hydroxychloroquine produced significant bias and/or instrument alarms. Overall, our study demonstrates that urine concentrations of hydroxychloroquine can reach very high concentrations (exceeding 500 mg/L) following overdose, with the potential to interfere with a range of urine assays including drug of abuse screening and microalbumin. Similar to previous reports, hydroxychloroquine overdose can produce hypokalemia and electrocardiographic abnormalities.