Background: Most of the available antimicrobial drugs have developed resistance; some of them suffer from severe toxicity, side effects. So, there is a need to discover novel compound(s) which should not only be potent, but also less toxic and cost effective.

Background: Method development, validation is an important parameter for the simultaneous estimation of Serratiopeptidase (SERA), Aceclofenac (ACE) and Paracetamol (PCM) by RP-HPLC, is supposed to be a costly and tedious process. The present study revealed using cheap and cost effective solvent system for the simultaneous estimation of Serratiopeptidase (SERA), Aceclofenac (ACE) and Paracetamol (PCM).Objective: Development and validation of a new chromatographic method for the simultaneous estimation of Serratiopeptidase (SERA), Aceclofenac (ACE) and Paracetamol (PCM) by RP-HPLC of marketed formulations.Methods: Simultaneous determination of SERA, ACE and PCM were carried out by RP-HPLC at the wavelength 327 nm, flow rate 0.4 mL/min, and the mobile phase used was water: methanol in the ratio (50:50 v/v). Further validation parameters such as system suitability, linearity, accuracy, precision, specificity, LOD, LOQ and robustness were taken into account to carry out the validation of the method. Results:Absorbance maxima for the simultaneous determination were selected by the UV spectrophotometer and that was found to be 327 nm in methanol and water. During the process of RP-HPLC, the linearity was obtained in the concentration range of 2-10 µg/mL for SERA, 100-500 µg/mL for ACE and 20-100 µg/mL for PCM. Correlation coefficient (r) for SERA, ACE and PCM in methanol and water was found to be 0.9817, 0.991 and 0.9949 respectively. Conclusion:The RP-HPLC method was simple, accurate, precise, and rapid and can be used for the simultaneous determination of SERA, ACE and PCM in bulk and pharmaceutical dosage form. The method is also economical as RP-HPLC grade water methanol in a ratio of (50:50) was used to achieve all the validation parameters.