The majority of fatalities from poisonous mushroom ingestion are caused by amatoxins. To prevent liver failure or death, it is critical to accurately and rapidly diagnose amatoxin exposure. We have developed a liquid chromatography tandem mass spectrometry method to detect α-, β-, and γ-amanitin in urine to meet this need. Two internal standard candidates were evaluated, including an isotopically labeled 15N10-α-amanitin and a modified amanitin methionine sulfoxide synthetic peptide. Using the 15N10-α-amanitin internal standard, precision and accuracy of α-amanitin in pooled urine was ≤5.49% and between 100 and 106%, respectively, with a reportable range from 1–200 ng/mL. β- and γ-Amanitin were most accurately quantitated in pooled urine using external calibration, resulting in precision ≤17.2% and accuracy between 99 and 105% with calibration ranges from 2.5-200 ng/mL and 1.0–200 ng/mL, respectively. The presented urinary diagnostic test is the first method to use an isotopically labeled α-amanitin with the ability to detect and confirm human exposures to α-, β-, and γ-amanitin.