Guingona was first elected to the Senate in 1987 under the Aquino-backed Lakas ng Bayan coalition. He was elected as Senate President pro tempore in 1987 and Majority Leader in 1990.

Additionally, he served as director and chairman of the Mindanao Development Authority and the Mindanao Labor Management Advisory Council.

In 1992, he ran for reelection under the Laban ng Demokratikong Pilipino of Speaker of the House Ramon Mitra, Jr. and won, placing 14th in the Senate race. He became Majority Leader again in 1993 but his term in the Senate was cut short when President Fidel V. Ramos appointed him as Executive Secretary the same year.

In 1998, he was elected again to the Senate under the Lakas-NUCD and was elected as Minority Leader. He boldly spoke out against the anomalies in the administration of President Joseph Estrada and was among the first to call for his resignation. Guingona exposed the graft and corruption, plunder, culpable violations of the Constitution, and cronyism in the Estrada administration that led to the impeachment trial of Estrada by the Senate. On January 17, 2001, he was one of the senators who voted in favor of opening an envelope that was said to contain incriminating evidence against Estrada. The final vote was 11-10, in favor of keeping the envelope closed, which further fueled anti-Estrada sentiments that led to another uprising in EDSA. When Estrada was ousted, Guingona emerged as the top choice for a successor to Vice President Gloria Macapagal-Arroyo, who succeeded Estrada as president.