Matrix-assisted laser desorption ionization time-of-flight mass spectrometry has been widely used as an emerging technology for the rapid identification of microorganisms.Cronobacter sakazakii (C. sakazakii)is a food-borne pathogen of particular importance to the powdered infant formula (PIF) processing environment due to its high lethality in infants. However, the traditional solid spotting detection method of pretreating samples for MALDI-TOF MS leads only to qualitative detection ofC. sakazakii. We developed a new, low-cost, robust liquid spotting pretreatment method and used a response surface methodology to optimize its parameters. The applicability, accuracy, and quantitative potential were measured for different types of samples. The optimal parameters of this method were as follows: a volume of 70% formic acid of 25 μL, treatment with ultrasound at 350 W for 3 min, and a volume of acetonitrile added of 75 μL. These conditions led to the highest identification score forC. sakazakii(1926.42 ± 48.497). This method was found to detect bacteria accurately and reproducibly. When 70 strains ofC. sakazakiiisolates were analyzed with this method, the identification accuracy was 100%. The detection limit ofC. sakazakiiin environmental and PIF samples was 4.1 × 101cfu/mL and 2.72 × 103cfu/mL, respectively.