The objective of this study was to find an efficient, rapid, simple, and cost-effective method of pretreating raw milk samples to produce PCR-ready DNA for subsequent microbial detection using the strains of eight bacterial species. A total of 17 in-house protocols and three commercial kits were evaluated in three steps from scientific, practical, and economic perspectives. The results showed that an in-house procedure involving Triton X-100-based pretreatment and an inhibitor removal resin was superior to all other methods tested in terms of DNA yield, sensitivity, ease of sample handling, time efficiency, and cost per sample. Overall, this simplified preanalytical protocol was shown to have a great potential for use in rapid detection of dairy-related bacterial species, thereby enabling early intervention in the food chain and thus reducing the risk of negative economic and health outcomes.