After shakedown in Long Island Sound, Grouper sailed for Pearl Harbor on 30 March 1942 to join the Pacific Submarine Force, which was to play havoc on Japanese shipping.

Before departing for her first war patrol, Grouper was assigned to the submarine screen which ringed the area as the American and Japanese fleets clashed in the decisive Battle of Midway.

Patrolling the fringe of the fighting on 4 June, Grouper sighted two burning enemy aircraft carriers, but could not close for attack because of heavy air cover.

On that day, she was strafed by fighter planes and driven deep in a series of aircraft and destroyer attacks which saw over 170 depth charges and bombs dropped on the novice submarine.

In addition to her regular patrol duties, which harassed Japanese shipping and tied up valuable warships desperately needed by the enemy, Grouper landed 50 men and 3,000 lb (1,400 kg) of gear on New Britain to carry on guerrilla warfare; at the same time, she rescued an American aviator who had been stranded there almost three months.

On 30 July 1943, a United States Army Air Forces B-25 Mitchell bomber mistakenly attacked Grouper in the Coral Sea, 185 nautical miles (343 km; 213 mi) east of Rossel Island at 11°00′S 156°44′E / 11.000°S 156.733°E / -11.000; 156.733.

She returned to Pearl Harbor on 6 August, but V-J Day cancelled plans for another patrol, and on 9 September, Grouper, in company with Toro and Blackfish, sailed for New London.

These operations ended 5 March 1950 as Grouper entered the Mare Island Naval Shipyard for conversion under project SCB 58 to the Navy's first "hunter-killer submarine".

With the addition of a snorkel and extensive sonar and radar facilities, Grouper emerged from the yard on 27 June 1951 to pioneer in research on the deadly submarine-versus-submarine warfare.

Her forward torpedo room was converted into a floating laboratory; work benches and additional berths for scientists were installed, and various types of sonar gear were added topside.

Her duties as a floating laboratory took her frequently to the Caribbean and Bermuda, although she retained New London as her home port and engaged in operations there and as far north as Nova Scotia.