High performance thin layer chromatography (HPTLC) method has been developed and validated for simultaneous estimation of Diclofenac Potassium and Serratiopeptidase. The separation was achieved on precoated silica gel 60 F254 aluminum plate as stationary phase and n-Hexane: Toluene: Methanol (6.0: 2.5:1.5 v/v/v) as mobile phase. The densitometric scanning was carried out at 260 nm. The Rf values were found to be 0.2183±0.0040 and 0.5966±0.0051 for Diclofenac Potassium and Serratiopeptidase respectively. Beer’s law obeyed in concentration range of 100-500 ng/spot and 40-200 ng/spot in methanol for Diclofenac Potassium and Serratiopeptidase respectively. The method was validated for specificity, linearity, accuracy, precision, LOD and LOQ. The percent assay of marketed formulation was 100.35±0.5379 and 99.93±0.7637% (2061.90 units/mg) of Diclofenac Potassium and Serratiopeptidase respectively. The proposed methods are simple, accurate and precise since they are recommended for routine analysis.
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