Opposition leaders have been locked in a standoff with President Viktor Yanukovych over his decision to scrap key political and free trade agreements with the European Union last month.
"The United States expresses its grave concern over an emerging pattern of targeted violence and intimidation towards activists and journalists who participated in or reported on the EuroMaidan protests," State Department spokeswoman Jen Psaki said in a statement.
"The violent beating of journalist Tetyana Chornovil is particularly disturbing," she said.
Chornovil, who writes for the Ukrainska Pravda opposition website, was attacked overnight Tuesday outside the capital Kiev, police said in a statement citing the journalist, who described being pulled from her car, beaten on the head and thrown into a ditch.
The attack came after a local pro-EU activist was stabbed in both thighs in the eastern city of Kharkiv on Tuesday evening.
The United States called on Ukraine to "ensure respect for human rights, including fundamental freedoms of expression and assembly, the rule of law, and democratic principles," Psaki said.
"We urge the government of Ukraine to send an unequivocal message that violence against critics of the government and those who are working towards a modern, democratic, and prosperous Ukraine will not be tolerated," she added, emphasizing that the US and its European allies would be watching closely.
Yanukovych's decision to scrap the EU pact sparked the largest protests since the pro-democracy Orange Revolution in 2004 but the demonstrations have been losing their momentum following a bailout deal with Russia last week.
Protesters have been occupying Kiev's central Independence Square,known locally as Maidan, since late November but opposition leaders have been unable to shake Yanukovych from his perch.