Simple SummaryDog cloning requires in vivo matured recipient oocytes to transfer somatic donor cells for somatic cell cloning. The timing for recovering in vivo matured dog oocytes was determined by predicting the ovulation day based on serum progesterone (P4) concentration in estrus bitches. Radioimmunoassay (RIA) is traditionally used to measure the P4 concentration in dogs. In this study, the P4 concentration for ovulation was measured using next-generation enzyme-linked fluorescence assay (ELFA) and it was compared with that measured using RIA. The oocytes collected in vivo showed a maturation rate of 65.19% after the prediction of ovulation based on the P4 range measured using the ELFA system. These oocytes then produced four cloned puppies. Conclusively, we provide the optimal P4 range for the prediction of ovulation in estrus bitches when ELFA is used to measure the P4 concentration. Thus, we prove the effectiveness of application of ELFA in obtaining in vivo matured oocytes for dog cloning.AbstractSuccessful dog cloning requires a sufficient number of in vivo matured oocytes as recipient oocytes for reconstructing embryos. The accurate prediction of the ovulation day in estrus bitches is critical for collecting mature oocytes. Traditionally, a specific serum progesterone (P4) range in the radioimmunoassay (RIA) system has been used for the prediction of ovulation. In this study, we investigated the use of an enzyme-linked fluorescence assay (ELFA) system for the measurement of P4. Serum samples of estrus bitches were analyzed using both RIA and ELFA, and the measured P4 values of ELFA were sorted into 11 groups based on the standard concentration measured in RIA and compared. In addition, to examine the tendency of changes in the P4 values in each system, the P4 values on ovulation day (from D − 6 to D + 1) in both systems were compared. The ELFA range of 5.0–12.0 ng/mL was derived from the RIA standard range of 4.0–8.0 ng/mL. The rates of acquired matured oocytes in RIA and ELFA were 55.47% and 65.19%, respectively. The ELFA system successfully produced cloned puppies after the transfer of the reconstructed cloned oocytes. Our findings suggest that the ELFA system is suitable for obtaining in vivo matured oocytes for dog cloning.