Mick McCarthy insists he will not tolerate any further shows of disrespect from Jay Bothroyd.
The Wolves striker angered McCarthy by gesturing to his manager after scoring in Saturday's 3-2 friendly defeat to Reading.
Bothroyd was immediately substituted and, while saying the matter has been put to bed, McCarthy confirmed he does not want to witness a repeat.
"I won't have it," McCarthy told the club's official website.
"Jay said he didn't gesture to me, but I felt it was aimed in my direction. I'll not have that level of disrespect.
"I treat people with respect and I expect that back.
"I don't care where we are in the League, how many points we've got or what my circumstances are, I will not accept that from anyone. But it's over now."
Meanwhile, Wolves keeper Matt Murray will miss the start of the season after being booked in for a knee operation.
Murray is expected to be out for at least three weeks, with McCarthy due to enter the transfer market to bring in a replacement.