The proposed method describes a high performance liquid chromatographic method with fluoremetric detection for the determination of cisatracurium (CIS) and propofol (PRP) simultaneously, which are co-administered as a pre-operative injection mixture. The separation of the two drugs was achieved using monolithic column (100 mm and 4.6 mm internal diameter) and mixture of methanol and 0.1 M phosphate buffer in the ratio of 80:20 (v/v) at pH 4.5 as a mobile phase. The fluorescence detection was carried out at 230/324 nm. The procedure showed good linearity through the concentration ranges of 0.01-1.00 μg/mL and 0.1-3.0 μg/mL with limits of detection of 0.002, 0.030 μg/mL and limits of quantification of 0.006, 0.100 μg/mL for CIS and PRP, respectively. Simultaneous determination of CIS and PRP in spiked human plasma samples was additionally executed and the results were satisfactory precise and accurate.