Routine hormone analysis is necessary for establishing and monitoring patient diseases. For example, the continuous monitoring of cortisol levels can help diagnose a patient with Cushing disease (high cortisol) or Addison disease (low cortisol). A robust method for steroid hormone determination in serum is imperative in diagnostics and treatment. XTR tips with 3 mg of Supel™ Swift HLB sorbent chemistry were used in Hamilton format. This allowed for reduced sample volume, sample evaporation mitigation, and offered a fully automated approach. In this method, a total of 9 steroids (cortisone, cortisol, 11-deoxycortisol, androstenedione, testosterone, dehydroepiandrosterone, 5-dihydrotestosterone, 17-hydroxyprogesterone, and progesterone) were analyzed as a panel to cover a variety of testing applications and disease diagnostics.