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P-glycoprotein (P-gp) is an efflux transporter of xenobiotic and endogenous compounds across the blood-brain barrier (BBB). P-gp plays an essential role by limiting the passage of these compounds into the brain tissue. It is susceptible to drug-drug interactions when interacting drugs are coadministered. The efficiency of P-gp may be affected by the aging process and the development of neurodegenerative diseases. Studying this protein in older adults is therefore highly relevant for all these reasons. Understanding P-gp activity in vivo is essential when considering the physiological, pathophysiological, and pharmacokinetic perspectives, as these aspects seem to be interconnected to some extent. In vivo exploration in humans is based on neuroimaging techniques, which have been improving over recent years. The advancement of exploratory and diagnostic tools is opening up new possibilities for understanding P-gp activity at the blood-brain barrier.