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Chlamydia trachomatis is an obligate human pathogen. It infects the genital tract of humans ascending into the fallopian tube, exacerbated by chronic pelvic pain, pelvic inflammatory disease, and fallopian tube scaring resulting in infertility and other malignancies. The major hurdle in controlling chlamydia l spread is that the infection remains asymptomatic, thus leading to chronic, recurrent and persistent infections, with no vaccines developed so far. Being a human pathogen, we do not have an in vivo model of C. trachomatis infection. C. trachomatis do not cause ascending infections and fallopian tube pathology in the mouse urogenital tract when infected vaginally. To overcome this hurdle trans cervical method of infection must be adapted. In this protocol the method of establishing trans-cervical Chlamydia l infection with the procedure to determine the bacterial load is detailed. This method will facilitate to deliver the bacteria past the cervix establishing an ascending infection into the uterine horns reciprocating human fallopian tube infections., Competing Interests: Competing interestsThe authors declare no competing interest., (Copyright © 2020 The Authors; exclusive licensee Bio-protocol LLC.)