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Dr. Gregory House is a maverick medical genius, who heads a team of young diagnosticians at the fictional Princeton-Plainsboro Teaching Hospital in New Jersey. Most episodes start with a cold open somewhere outside the hospital, showing the events leading to the onset of symptoms for that week's main patient. The episode follows the team in their attempts to diagnose the illness.

The team arrives at diagnoses using the Socratic method and differential diagnosis, with House guiding the deliberations. House often discounts the information and opinions from his underlings, assuming their contributions to miss relevant unconsidered factors. The patient is usually misdiagnosed two or three times over the course of each episode, almost always including such diseases as sarcoidosis, and treated with medications appropriate to those diagnoses that cause further complications. Often the ailment cannot be easily deduced because the patient has lied about symptoms and circumstances. House frequently mutters, "Everybody lies", or proclaims during the team's deliberations: "The patient is lying." Even when not stated explicitly, this assumption guides House's decisions and diagnoses.

House's begrudging fulfillment of his mandatory walk-in clinic duty is a recurring subplot on the show. During clinic duty, House confounds patients with an eccentric bedside manner and unorthodox treatments, but impresses them with rapid and accurate diagnoses after seemingly not paying attention. He often plays video games on his Nintendo Game Boy Advance SP while patients talk to him, and in one episode House diagnoses multiple patients in the waiting room in under five minutes on his way out of the clinic. Realizations made during some of the simple problems House often faces in the clinic help him solve the main case of the episode — ironic, because he claims to hate working in the clinic.

Episodes frequently feature the unusual practice of entering a patient's house with or without the owner's permission in order to search for clues that might suggest a certain pathology. The creator, David Shore, originally intended for the show to be a CSI-type show where the "germs were the suspects", but has since shifted much of the focus to the characters rather than concentrating solely on the environment.

An apparent in-joke in the series is that one condition, lupus, is suggested as a cause of the patient's symptoms in virtually every episode, although invariably this is quickly dismissed. One episode self-referentially features the line, "It's never lupus!"