Four simple, accurate and precise methods have been developed and validated for the determination of cisatracurium besylate in bulk and in its pharmaceutical preparation. The first method was a spectrofluorimetric method based on measuring the native fluorescence intensity (FI) of cisatracurium (CIS) in aqueous micellar medium at 317 nm upon excitation at 235 nm in the range of 0.2- 2.2 µg.mL-1. The other three methods were adopted for the determination of the studied drug in the presence of its alkaline degradation product including a spectrophotometric method namely, first derivative of ratio spectra (DD1) in the range of 8-38 µg.mL-1 and two high pressure liquid chromatographic (HPLC) methods, one with diode –array detector (DAD) and the other with fluorescence detector (FLD) in the ranges of 1.0- 40.0 µg.mL-1 and 0.25-20.0 µg.mL-1, respectively. Separation was achieved on Supelco Discovery®C18 column (150 mm x 4.6 mm, 5 µm) and Agilent Zorbax® SB- CN column (50 mm x 4.6 mm, 1.8 µm) for HPLC - DAD and HPLC- FLD methods, respectively. All the proposed methods were validated and successfully applied for the determination of CIS in bulk and in pharmaceutical preparation with good recovery ranges between 99.92- 100.71. The results obtained by applying the proposed methods were statistically analyzed and compared with those obtained by the manufacturer method, and no significant difference was found.