Background: The quality of multiplex polymerase chain reaction (PCR) assays depends on several factors.Therefore, it is important to establish the optimal conditions to achieve efficient amplification.The objective of this study was to implement a 5 × 4 factorial design combined with image analysis using agarose gels and an efficiency calculation to optimize a multiplex PCR assays for the detection of Salmonella enterica serovar typhimurium.Methods: We used 12 ng of Salmonella DNA obtained from pure cultures and applied different annealing temperatures (65 • C, 64.5 • C, 63.3 • C, 61.4 • C, or 59 • C) and different MgCl 2 concentrations (1 mM, 1.5 mM, 2 mM, or 2.5 mM) to amplify regions of the fliC, rfbJ, and fljB genes.The 5 × 4 factorial design was performed using Statgraphics Plus software version 5.1, and the images were analyzed using Image Lab TM software.Results: Superior amplification was obtained using an annealing temperature of 65 • C and 2 mM MgCl 2 .This finding was confirmed by calculating the efficiency of multiplex PCR assays (6.1%) at these conditions.Conclusion: We propose the application of factorial design and image analysis to determine the most suitable conditions for multiplex PCR optimization.