To obtain in vivo matured oocytes for dog cloning, serum progesterone (P4) level were employed for ovulate determination. Radioactive immunoassay (RIA) is a traditional serum hormone assay method with highly radioactivity. The aim of this study was to evaluate the reliability of RIA and to compare its canine serum P4 concentration determination accuracy to that of the electric chemiluminescence immunoassay (ECLI). To obtain in vivo matured oocytes for canine somatic cell nuclear transfer, serum P4 levels were accurately measured with both methods of RIA and ECLI. Although both methods detected similar P4 level before ovulation, the mean P4 concentration using ECLI was significantly higher than that using RIA from 3days before ovulation. Following ovulation, oocytes were collected by surgery, and a lower percentage of mature oocytes were observed using ECLI (39%) as compared to RIA (67%) if 4-8ng/ml of P4 were criteria for determination of ovulation. On other hand, high percentage of mature oocytes was observed using ECLI when 6–15 ng/mL of progesterone was criteria for ovulation determination. To determine whether in vivo oocytes obtained by ECLI method could be used for canine cloning, six canines were selected as oocyte donors and two puppies were produced after SCNT and embryo transfer. In conclusion, compared to the traditional RIA method, the ECLI method is a safe and reliable method for canine cloning.