The Spanish capital club had two penalty claims waved away by the referee for fouls on Cristiano Ronaldo as they were unable to beat Valencia goalkeeper Guaita, who was the man of the match.
"The refereeing? 80,000 people saw we were persecuted, did they not?," Karanka told reporters at a press conference.
"Because if we say it, then it all kicks off. Everybody saw it."
Madrid are now only four points clear at the top of the Liga table, after Barcelona defeated Zaragoza 4-1 on Saturday, but Karanka says that his side are as determined to win La Liga as ever.
"We are calm because we are four points above Barcelona," Karanka said. "I know the atmosphere in the dressing room and we are calm.
"We are thinking about our next match and we have players who are eager to continue to fight to win La Liga."
Los Blancos will take on Atletico Madrid at the Vicente Calderon on Wednesday in their upcoming Liga fixture.