Bacteria are ubiquitous in the environment and may cause disease and mortality in cultured shrimp and fish both in the hatchery and grow-out ponds. Accordingly, antimicrobial agents are widely used by farmers especially in the intensive culture system. Misuse of drugs and non-compliance of treatment regimens among users can cause treatment to be less effective and prolong the duration of disease. Furthermore, they may increase the prevalence of transferable resistance genes to the bacteria in the environment and in food fish that may indirectly affect human health due to limited therapeutic options. Since several antimicrobial agents are widely used in various areas of shrimp and fish culture, testing for the susceptibility and resistance of bacteria to different agents need to be conducted. The data gathered from the tests are essential in the selection of the most effective antimicrobial agents against pathogenic bacteria. Furthermore, the values obtained in the tests are useful in determining the dosage regimen of each antimicrobial. Different methods have been employed in laboratories to determine the susceptibility of microorganisms to antimicrobial agents. Two main methods for the in vitro determination of the susceptibility of microorganisms against antimicrobials are the disk agar diffusion test, wherein antibiotic-impregnated disks are used with an agar medium, and the dilution techniques, wherein the test microorganism is exposed to increasing concentration of an antibiotic either in broth or agar. The disk agar diffusion method is the most widely used laboratory technique for antimicrobial susceptibility test. This method is simple, requires a short duration, and easy to perform. It also provides an accurate determination of the susceptibility of an organism to a measured amount of test antibiotics. However, only a few antimicrobials have veterinary-specific interpretative criteria; therefore, human interpretative criteria are used for the majority of the chemotherapeutants. Human interpretative criteria could, however, vary from veterinary-specific data due to strain differences in the bacterial species. The dilution technique, using broth and agar media, is used to measure quantitatively the in vitro activity of antimicrobial agents in terms of susceptibility and resistance by bacteria (Thornsberry et al., 1977; Thornsberry and McDougal, 1983). The minimal inhibitory concentration (MIC) using agar dilution is one of the appropriate in vitro tests widely used among several laboratories (McDonald et al., 1995). The results obtained by MIC dilution test can give an indication as to the concentration of antimicrobial agent needed to inhibit or eradicate the infectious organism. Basically, a series of plates is prepared with an agar medium to which various concentrations of the antimicrobial agents are added. The plates are then inoculated with a suitable standardized suspension of the test bacteria. After the plates are incubated overnight under a certain temperature, examination of the test results is performed, MIC values are read, and drug resistance of the bacterial strains is determined. In both techniques, it is important that control strains be included to check the quality of batches of media and to monitor the performance of the assay protocol. The selection of the control strain should be based on the kind of bacteria being investigated in any set of experiments. The National Committee for Clinical Laboratory Standards (NCCLS) recommended the use of the following organisms for the quality control of antimicrobial susceptibility test: Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis, Actinobacillus pleuropneumoniae, Haemophilus somnus, Campylobacter jejuni, Streptococcus pneumoniae, and Klebsiella pneumoniae. The series of procedures, materials preparation, as well as the interpretative criteria to obtain the final results in antimicrobial sensitivity testing are influenced greatly by the method used, which must be standardized and carefully controlled. The standard methods found in this manual are written for the students, teachers, laboratory technicians, researchers, and scientists who work in this field.