Agrawal said Twitter's estimate, which has stayed the same since 2013, cannot be reproduced externally given the need to use both public and private information to determine whether an account is spam.
Musk, who on Friday said his $44 billion deal to buy Twitter was "temporarily on hold" pending information on spam accounts, responded to Agrawal's defence of the company's methodology with a poop emoji.
"So how do advertisers know what they're getting for their money? This is fundamental to the financial health of Twitter," Musk wrote.
Shortly after his tweets, Musk told a private conference in Miami that he suspects bots -- or automated accounts -- make up about 20 percent to 25 percent of users, according to tweets by attendees.
Twitter shares on Monday fell below levels preceding Musk's disclosure of a 9.2 percent stake in the company in early April.
The shares were down 7.7 percent, at $37.50 a share, in afternoon trade. That compares to a closing price of $39.31 on April 1, the last trading day before Musk disclosed his stake.
Musk has pledged changes to Twitter's content moderation practices, railing against decisions like the company's ban of former President Donald Trump as overly aggressive while pledging to crack down on "spam bots" on the platform.
Musk has called for tests of random samples of Twitter users to identify bots, and said he has yet to see "any" analysis that shows spam accounts making up less than 5 percent of the user base.
Musk said on Sunday "there is some chance it might be over 90% of daily active users."
Independent researchers have estimated that anywhere from 9 percent to 15 percent of the millions of Twitter profiles are bots.
Twitter does not currently require users to register using their real identities, and automated, parody and pseudonymous profiles are expressly permitted on the service.
It does ban impersonation and spam, and penalises accounts when the company determines their purpose is to "deceive or manipulate others" by engaging in scams, coordinating abuse campaigns or artificially inflating engagement.