This study aimed to measure the concentration of progesterone (P4) in pregnant, mated/non-pregnant, and anestrous controlled Awassi ewes as well as in ewes carrying singletons and twins. A total of 35 ewes were used, which were subdivided into two groups: control (5 ewes) and treated (30 ewes). The estrus was synchronized in the treated group using P4-intravaginal sponge with eCG. All ewes were examined by ultrasound in different periods to confirm pregnancy status and fetal number. Blood samples were collected from ewes in both groups at four different time dates (15, 25, 35, and 50 days) post-insemination (PI). Results showed that P4 levels were significantly superior (P<0.05) in pregnant (ranging from 4.90±0.21-7.55±0.23 ng/mL) compared to mated/non-pregnant ewes (ranging from 2.52±1.62-1.112±1.2 ng/mL) and control ewes (ranging from 0.53±0.15-0.579±0.3ng/ mL) during all four experimental periods. Additionally, the P4 concentrations of ewes with twin fetuses were significantly (P<0.05) elevated compared to ewes carrying only one fetus at days 35 (7.36±0.24 Vs 5.68±0.11) and 50 (8.89±0.13 Vs 7.36±0.24) PI, but not significantly before that. This study concluded that a threshold value of >4.9 ng/mL can be used to differentiate between pregnant and empty ewes from day 25 onwards. The human P4 kit was precise and showed significant differences between pregnant, mated/non-pregnant, and anestrous ewes (control) during all experimental periods. Furthermore, P4 concentrations were detected to be significantly higher in twin-bearing ewes compared to a singleton at day 35 PI and beyond.