ABSTRACT Background: Measurement of free testosterone (T) concentrations is indicated in the diagnosis of androgen disorders, including hypogonadism in men; hirsutism, polycystic ovary syndrome (PCOS), and androgenic alopecia in women; pubertal disorders in boys and management of gender affirming hormone therapies for transgender and gender diverse (TGD) persons. This Phase IIB proposal aims to continue the development of the TruTTM algorithm by validating it in common conditions characterized by altered estradiol (E2), T, and SHBG concentrations and incorporating interaction of E2 with T for wider commercial adoption in women in whom E2 levels vary greatly across the menstrual cycle and in TGD population. Approach: This application follows the FDA’s published “Guidance for Industry: Bioanalytical Method Validation”. The essential parameters to determine the acceptability of a bioanalytical method include its technical performance (accuracy, precision, sensitivity, selectivity, stability, and matrix effects). Reference ranges should be determined in appropriate human samples. The analytical method should be validated for intended use (e.g., determination in conditions of intended use, such as persons with altered E2 and T levels, women with PCOS, TGD persons etc.). In studies through the Phase II, we demonstrated that the method has superior performance characteristics and extended the validation of TruTTM algorithm in conditions characterized by altered SHBG concentrations. In the proposed Phase IIB studies, we will generate the v2.0 of TruTTM algorithm by incorporating the dynamics of the E2 induced perturbation in free T levels, validate it in men, women and TGD populations (Aim 1) and deploy HIPAA compliant, secure integration of the algorithm into electronic medical records (EMR) workflow (Aim 2). Future Directions and Commercialization potential: The phase IIB program will enable the pilot commercial deployment of a HIPAA compliant (FDA registered) platform for commercializing the TruTTM (v2.0) algorithm embedded into electronic medical record (EMR) for wider clinical adoption. These studies will improve clinical care and advance our fundamental understanding of dynamic regulation of T bioavailability in diverse populations including unrepresented sexual and gender minorities.