Antibiotics are used as prophylactic agents to promote growth and for treating infections in animals. However, the irrational use of antibiotics in livestock management is a significant cause of the development of antibioticresistant genes in the environment. Each year 2 million people suffer from the infections caused by bacteria which are resistant to antibiotics and 23,000 of these people are estimated to die because of antibiotic resistance. New drugs are continually coming into the market but are at the risk of developing resistance. Thus, there is a need for the development of analytical methods which can be used to monitor these antibiotic concentrations in environmental samples. This research is focused on developing and validating a Solid Phase Extraction (SPE) procedure and liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying tylosin antibiotic in cattle waste. Tylosin was extracted from cattle waste samples using Strata polymeric weak cation cartridges by adding a sodium-EDTA buffer solution and methanol. Chemical analysis of the extracted tylosin was performed using a Varian 212-LC HPLC and Agilent 500 Ion Trap mass spectrometric detector. The concentrations of tylosin in study group animals were compared with respect to the date of sampling and cattle body weight with a control group and results are presented.