Background/ Ethnopharmacological Relevance\nAyurveda, the system of traditional medicine native to the India and a form of alternative medicine, emphasizes the use of cow’s urine as an effective medicine against various skin diseases, over the centuries. A complete biochemical analysis of cow’s urine was done in this research, to understand its biochemical constituents, including tests to confirm the presence of phenols, flavonoids, antioxidants and steroids.\nMaterials and Methods\nA set of quantitative and qualitative tests that baselined methodologies/ estimations using Gallic acid for phenols, vanillin-catechol for flavonoids, 2,4-dinitrophenyl hydrazine (DNPH) for antioxidants, etc. were applied. The antioxidant activity was assessed by hydroxyl radical scavenging activity using DNPH reagent. Diastase enzyme activity was calculated in terms of maltose equivalent using DNS reagent estimation method. HPLC analysis was done using Gallic acid standard. Hydrocortisone steroid estimation using 2,4-dinitrophenyl hydrazine was also done successfully.\nResults\nThe research highlights cow’s urine as a substantial source of natural antioxidant, helpful in preventing the progress of various oxidative stresses. Hydrocortisone estimation confirms cow’s urine to be an excellent natural source of steroids too, and ascertains its wide array of medical applications as a natural source of antioxidants, steroids, and other enzymes.\nConclusions\nThis research ascertains the pharmaceutical applications of cow’s urine, being a natural source of significant bioconstituents with no harmful side-effects and cost-effectively cheaper in extraction/ purification processes.