Aquaculture production has been sharply increasing in Brazil in the last years. Many classes of antimicrobials are commonly used in aquaculture worldwide to treat infections caused by a variety of bacterial pathogens and they are also used as a growth promoter. However, these intense uses may cause environmental contamination and bacterial resistance. A method was developed and validated for simultaneous assessment of 12 drugs of different antimicrobial classes (chloramphenicol, florfenicol, oxytetracycline, tetracycline, chlortetracycline, sulfadimethoxine, sulfathiazole, sulfamethazine, enrofloxacin, ciprofloxacin, norfloxacin, and sarafloxacin) on the Nile tilapia’s muscle (Oreochromis niloticus). This study presents the development of a rapid method using ultrafiltration by Captiva cartridges and liquid chromatography tandem mass spectrometry triple quadrupole (Agilent 6430 – Agilent Technologies) in a negative mode for florfenicol and a positive mode for the others. The sample pretreatment involves extraction with 5 g of muscle fish, 1 mL of 0.1 M Na₂EDTA and 24 mL of acetonitrile: water (0.1 % formic acid; 70:30), and purification by Captiva cartridges, followed by the determination of all compounds in a single run. Sulfadimethoxine-d6 was used as an internal standard to obtain more reliable results. The developed method was validated based on Eurachem Guide: The fitness for purpose of analytical methods, with the calibration curves carried out at blank samples spiked, matrix-matched calibration (MMC). The limits of quantification were lower than 4.3 μg kg^-1 for all compounds; calibration curve showed linearity at the work range, recovery ranged from 83.8% to 110.1%, and accuracy was lower than 5.5%. The developed analytical method was successfully applied in 36 fish samples collected in 4 fish farms in the most important producing region of São Paulo State, Brazil.