c"It's either Rafah or the hostages - choose life," reads a banner waved by protesters as they blocked Tel Aviv's Ayalon Highway in both directions, the public broadcaster KAN reported.
Two people, including a woman, were arrested for attempting to move the protesters away.
Reports have emerged of a cease-fire proposal being discussed by Israel and Hamas to end the conflict in the Gaza Strip.
Israeli Prime Minister Benjamin Netanyahu, however, has vowed to invade Rafah, home to more than 1.4 million displaced Palestinians, with or without a deal with Hamas.
Hamas, which is believed to be holding more than 130 Israeli hostages, demands an end to Israel's deadly offensive on the Gaza Strip in return for any hostage deal with Tel Aviv.
Israel has waged an unrelenting offensive on the Palestinian enclave since a cross-border attack by Hamas last Oct. 7 which killed some 1,200 people.
Nearly 34,600 Palestinians have since been killed, mostly women and children, and thousands of others injured amid mass destruction and severe shortages of necessities.
More than six months into the Israeli war, vast swathes of Gaza lay in ruins, pushing 85% of the enclave's population into internal displacement amid a crippling blockade of food, clean water and medicine, according to the UN.
Israel stands accused of genocide at the International Court of Justice. An interim ruling in January ordered Tel Aviv to stop genocidal acts and take measures to guarantee that humanitarian assistance is provided to civilians in Gaza.