A method to accurately and quickly determine milk-derived ingredients in infant formula was developed by the combined use of a genetic detection method for rapid screening and two-dimensional electrophoresis for confirmation, and its sensitivity and accuracy were evaluated. The one-step DNA extraction method based on thermophilic protease allowed the obtainment of DNA just through a temperature-controlled reaction within 17 min, which greatly reduced the sample pretreatment time. The fast real-time polymerase chain reaction (real-time PCR) program took only 28 minutes and 26 seconds. The limit of detection of the method was 10–100 pg/μL DNA for cow, buffalo, yak milk-derived ingredients using the universal primer, for goat and sheep milk-derived ingredients using the universal and specific primers as well as for mammalian milk internal reference, while the sensitivity was 1% (m/m) for bovine milk-derived ingredients in mixed samples. Then the samples that tested positive for bovine genes were examined by two-dimensional electrophoresis for whether they contained bovine caseins or whey proteins in order to determine their authenticity. The combination of the two methods can solve the bottleneck problem of difficult identification of milk-derived ingredients in infant formula by conventional testing methods, due to the complex ingredient composition of infant formula.