Background: Ayurveda, an ancient system of medicine, emphasizes holistic well-being through the balance of doshas and Dhatus. Dhatu Sarata assessment, a critical component in Ayurveda, evaluates tissue strength and quality, guiding personalized treatment strategies. Existing questionnaires for Dhatu Sarata assessment lack standardization and validation, necessitating the development of a reliable tool. Aim: To develop a standard and valid questionnaire for Dhatu Sarata assessment and create user-friendly software for its administration. Objectives: The study aimed to examine available questionnaires, review relevant literature, study classical Ayurvedic texts, consult with subject and statistics experts, and collaborate with software designers to develop and validate a comprehensive Dhatu Sarata assessment tool. Methods: A comprehensive literature review of existing questionnaires and Ayurvedic classics (Brihatrayee, Laghutrayee, Yogratnakar, Bhela Samhita, Harita Samhita, Kashyap Samhita) was conducted. Expert consultations were held to refine the questionnaire. Statistical analysis, including the unpaired t-test, was employed to validate the tool. An Android application was developed and tested for usability and accuracy. Results: Observations from 145 samples revealed significant differences between manual and app-based Dhatu Sarata assessments. The app consistently underestimated Sarata values compared to manual methods, with significant discrepancies in Rasa, Rakta, Mamsa, Meda, Asthi, Majja, Shukra, and Satwa Sarata (p<0.05). Discussion: The significant differences highlight the need for further refinement of the app's algorithms to align with traditional manual assessments. The app's potential for scalability and efficiency in clinical settings underscores the importance of ongoing development and validation. Conclusion:The study successfully developed a questionnaire and Android application for Dhatu Sarata assessment. While the app requires further refinement to ensure accuracy, it represents a significant advancement in integrating Ayurvedic practices with modern technology. Future research should focus on iterative testing and refinement to enhance the tool's reliability and applicability. Keywords: Ayurveda, Dhatu Sarata, questionnaire, software development, validation, traditional medicine, holistic health.