There was considerable doubt about whether the match would be played due to the civil unrest in Ukraine, where the contest was originally suppose to take place.
But after discussions it was decided to move the game to Cyprus, where goals from Andriy Yarmolenko and Marko Devic helped Ukraine claim the win.
The American defense struggled throughout the match and watched as Denis Garmash made a well-timed run through the middle of the back line in the 12th minute.
He brought the ball down inside the area and had his shot saved by goalkeeper Tim Howard. However, he collected the rebound and squared the ball to Yarmolenko, who easily finished from 10 yards.
Garmash had the ball in the net again for Ukraine five minutes later, but the goal was ruled out for offside.
The Americans came close to equalizing in the 51st minute when a header from Jozy Altidore sailed just over the crossbar following a right-wing cross from Geoff Cameron.
However, U.S. the back line fell apart again in the 67th minute as a flicked header from Roman Bezus released Devic in behind the defense.
His initial effort was saved by Howard, but he rolled home the rebound to double the advantage.
U.S. substitute Aron Johannsson had a volley cleared off the goal line by a Ukrainian defender in the closing minutes to prevent the Americans from finding the net, and they will next face Mexico in a friendly on April 2.