This chapter presents a description of Rocuronium bromide, which is a non-depolarizing neuromuscular blocking agent that produces rapid muscle relaxation after being injected intravenously. Furthermore, it is an aminosteroid neuromuscular blocking agent that has a propenyl (-CH2-CH1/4CH2) group on its active quaternary nitrogen atom. The compound is pharmacologically used to provide muscle relaxation in general anesthesia for surgical procedures and to aid in controlled ventilation. In the market, Rocuronium bromide is only available as a solution dosage form intended for intravenous or intramuscular injection. The determination of Rocuronium bromide in biological samples is done by liquid chromatography, thin-layer chromatography, and gas chromatography. The main advantage of using a fluorimetry detector is its inherently high sensitivity for pharmaceutical analysis, but very often application of this method to non-fluorescent drug substances requires derivatization with strong fluorophores. The derivatization process can be performed either pre-column or post-column to increase the detection sensitivity. The thin layer chromatography method was developed to confirm the identity of Rocuronium bromide and its metabolites prior to their determination using high-performance liquid chromatography (HPLC) coupled with fluorescence detection. Two gas chromatographic methods have been reported for the determination of Rocuronium bromide and its metabolites in biological samples.