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Ashwagandha is a medicinal plant that produces withanolides, important secondary metabolites with high medicinal value. Withanolides are predominantly found in the roots; therefore, improving root biomass is relevant for deriving greater benefits from this crop. Aeroponic technology offers a potential approach for maximising root production. However, growing plants in an aeroponic system requires a suitable nutrient solution and spray schedule, as these two components are crop specific. Therefore, the nutrient solution and spray schedule were standardised for ashwagandha plants. Accordingly, standard Hoagland's solution supplemented with an additional 50% N and 50% K, together with a spray schedule of 1 minute ON and 3 minutes OFF, was found to be effective in maximising plant growth. Following this protocol, plants were grown in aeroponic chambers. For comparison, plants of the same age were also grown in a root study structure in the field. When the plants were 120 days old, moisture stress was imposed in aeroponic chambers by reducing the spray frequency to 1 minute ON and 6 minutes OFF and in the field by withholding irrigation for 60 days to examine its effect on root morphology and secondary metabolite production. At maturity, both field-grown and aeroponically grown plants were harvested, and biometric parameters and secondary metabolite contents were measured. The results indicated that field-grown plants had thicker and sturdier roots with different root architecture than aeroponically grown plants, whose roots were thinner and longer. Field-grown plants also had significantly higher secondary metabolite contents, especially withanolides, than aeroponically grown plants under both control and stress conditions.