The aim of this study is to determine whether the serum concentrations of progesterone and/or prolactin after fresh embryo transfer are associated with pregnancy rates in in vitro fertilization treatment. This retrospective cohort study evaluated in vitro fertilization treatments with fresh embryo transfer, which were performed between 2013 and 2019 in a private clinic in Curitiba, Brazil. The serum concentrations of progesterone and prolactin were evaluated by chemiluminescence nine days after oocyte retrieval. The type of progesterone supplementation and pituitary blockage during the cycle of oocyte stimulation were evaluated. 330 fertilization cycles were performed in the 293 studied patients. The mean age of patients was 35.5±4.1years. The most seen isolated infertility factor was endometriosis (24.2% of the cases), while progesterone supplementation was performed intramuscularly in 73.9% of the cases. The progesterone values above 32.1ng/ml at day 9 (D9) were associated with better pregnancy rates. In cycles using antagonist and intramuscular luteal phase supplementation, higher pregnancy rates with progesterone values above 37.83ng/ml were observed. Moreover, prolactin showed no significant association with any of the studied variables. The serum progesterone concentrations above 32.1ng/ml at D9 that were taken one week before pregnancy testing were associated with successful in vitro fertilization treatment. Furthermore, prolactin showed no significant association with any of the studied variables.