In vaginal depositors, semen is deposited at the opening of the cervix, and sperm quickly enter the cervical canal, which is filled with cervical mucus in the lead up to ovulation. The outward flow of cervical mucus in the cervical lumen is a major challenge to sperm progression, and only a small percentage of the deposited sperm make their way into the uterus. Sperm–cervical mucus penetration tests are useful to assess the ability of both sperm and mucus to interact when in varying physiological states of the oestrous cycle. As sheep are vaginal depositors of semen, they are a useful model to study cervical sperm transport and thus ex vivo sperm–mucus penetration tests are often used by researchers developing products to manipulate cervical mucus to either block sperm or promote sperm transit across the cervix. Here, we describe a method to collect ovine cervical mucus, assess its quality, and perform a mucus penetration test using fluorescently tagged sperm which simplifies sperm counting.