Many art historians consider The Gross Clinic to be one of the best American paintings ever made. Dr. Samuel D. Gross, a seventy-year-old professor dressed in a black frock coat, lectures a group of Jefferson Medical College students. Included among the group is a self-portrait of Eakins, who is seen at the right-hand side of the painting, next to the tunnel railing, with a white cuffed sleeve sketching or writing. Seen over Dr. Gross's right shoulder is the clinic clerk, Dr. Franklin West, taking notes on the operation.

Admired for its uncompromising realism, The Gross Clinic has an important place documenting the history of medicine—both because it honors the emergence of surgery as a healing profession (previously, surgery was associated primarily with amputation), and because it shows us what the surgical theater looked like in the nineteenth century.

The painting is based on a surgery witnessed by Eakins, in which Gross treated a young man for osteomyelitis of the femur. Gross is pictured here performing a conservative operation as opposed to an amputation (which is how the patient would normally have been treated in previous decades).

Here, surgeons crowd around the anesthetized patient in their frock coats. This is just prior to the adoption of a hygienic surgical environment (see asepsis). The Gross Clinic is thus often contrasted with Eakins's later painting The Agnew Clinic (1889), which depicts a cleaner, brighter, surgical theater, with the participants in "white coats". In comparing the two, the advancement in understanding of the prevention of infection is seen. Another noteworthy difference in the later painting is the presence of a professional nurse, Mary Clymer, in the operating theater.

It is assumed that the patient depicted in The Gross Clinic was a teenage boy, although the exposed body is not entirely discernible as male or female; the painting is shocking for both the odd presentation of this figure and the matter-of-fact goriness of the procedure.

Adding to the drama is the lone woman in the painting seen in the middle ground, possibly the patient's mother, cringing in distress. Her dramatic figure functions as a strong contrast to the calm, professional demeanor of the men who surround the patient. This bloody and very blunt depiction of surgery was shocking at the time it was first exhibited.

Eakins's signature is painted into the painting, on the front of the surgical table.