Non-surgical transfer techniques for mouse embryos and sperm provide animal welfare benefits for assisted reproduction of mice. While surgical embryo transfer (ET) is an effective method for transfer of embryos into the uterine horn of mice, surgery is expensive, time consuming, and requires technical expertise. Surgery is also a stressful procedure for the mouse, which has to be anesthetized and treated with an analgesic. We have developed a simple, brief procedure for ET using a non-surgical embryo transfer (NSET) device. Once embryos are loaded into the NSET device, the tapered NSET catheter passes through the vagina and traverses the cervix to deposit the embryos into the uterine horn of a recipient mouse. Since the NSET procedure does not require sedation, opening of the inner body cavity, or use of an analgesic, this procedure is less stressful for the mouse than surgery. In proof, our results show that when pseudopregnant mice are monitored by electrocardiography, the non-surgical procedure does not affect heart rate. The NSET procedure also has no effect on the level of the stress biomarker fecal corticosterone measured by ELISA analysis. However, surgery or anesthesia does lower heart rate and increases levels of fecal corticosterone. Additional studies show that the NSET procedure is effective for transfer of mouse embryos for the purposes of rederivation, cryorecovery, and transgenic research. Embryos can be successfully transferred with the NSET technique after cryopreservation, in vitro fertilization (IVF), or embryonic stem (ES) cell injection as an alternative to traditional surgical embryo transfer. The NSET device has also successfully transferred mouse sperm for artificial insemination. Therefore, the NSET technique is an example of a 3Rs refinement, an alternative to a surgical procedure, which will lead the way for improved assisted reproductive techniques in mice.