This paper presents a new high performance liquid chromatography (HPLC) method for the identification and quantification of S-allyl-l-cysteine (SAC), which is the most important bioactive compound present in heated garlic juice (HGJ). This method uses LC combined with an ultraviolet (LC–UV) and mass spectrometry detection (LC–MS/MS) method and is based on the dansyl chloride derivatization step. The method validation included selectivity, linearity, precision, accuracy, and sensitivity. The linear correlation coefficients (r) were always greater than 0.998. Intra- and inter-day precision for SAC at three concentrations demonstrated a relative standard deviation (RSD) of less than 15.3% for both UV and MS methods. The limit of quantification (LOQ) was 0.71 μg/g and 0.07 μg/g for UV and MS methods, respectively. The results were not significantly different between the SAC contents determined by the UV and MS detection in real HGJ samples. Both methods are proposed as useful quality control tools for accurate determination of SAC content of garlic preparations.