Track and field athletes aiming to take part in the female category in elite competitions will have to take a gene test from September, World Athletics has announced.
The test will be in place in time for the World Championships which take place in Tokyo on September 13-21.
"It is really important in a sport that is permanently trying to attract more women that they enter a sport believing there is no biological glass ceiling," said World Athletics president Sebastian Coe.
"The test to confirm biological sex is a very important step in ensuring this is the case."
The test for the SRY gene, which is part of the Y chromosome and causes male characteristics to develop, can be done by a cheek swab or a blood test.
If the athlete tests negative for the Y chromosome they are eligible to compete in the female category in world ranking competitions.
If it is positive, they can only compete in the female category in non-world ranking competitions or in another category other than the female one.
The test will be administered by member federations.
World Athletics says the test is "extremely accurate", adding: "The risk of false negative or positive is extremely unlikely."
The SRY test is the same one adopted by World Boxing when it brought in mandatory sex testing for all competitors this year.
Coe said in a statement on Wednesday: "We are saying, at elite level, for you to compete in the female category you have to be biologically female.
"It was always very clear to me and the World Athletics Council that gender cannot trump biology."
The sport of athletics has long considered introducing eligibility criteria for women's events, amid questions over biological advantages for transgender athletes and those with differences of sex development (DSD).
Transgender women who have gone through male puberty are currently banned by World Athletics from competing in women's events, while the federation requires female DSD athletes whose bodies produce high testosterone levels to take medication to lower them in order to be eligible.