Critically ill patients often present with a combination of disease states and comorbid conditions that progress over a clinical course. This can manifest in physiological changes, such as fluid shifts, alterations in protein binding, and acid–base balance issues, which may in turn alter a drug's distribution, potentially towards or away from its site of action. It's vital that these factors are examined for drugs used in critical illness in varying disease states, acute and chronic in nature. Several methods have been used to study the variations in target site penetration, but few provide a feasible option to reliably measure active drug concentrations at the site of action over time. This review examines these techniques, their merits and shortcomings, generally and as they relate to use in critically ill.