The present study was designed to standardize protocol for asepsis of different explants of Alstroemeria hybrida cv. Pluto (rhizome tips, rhizome sections, shoot tips and shoot nodal segments). Carbendazim 200 ppm for 30 minutes + mercuric chloride (0.1%) dip for 10 minutes and final treatment with ethyl alcohol (70%) for one minute proved best for culture asepsis of rhizome tips and rhizome sections at 2 and 4 weeks of culture, whereas carbendazim 200 ppm for 15 minutes followed by mercuric chloride 0.1% dip for 3 minutes proved better for culture asepsis of shoot tip and shoot nodal segments at 2 and 4 weeks of culture. Also carbendazim 200 ppm for 30 minutes + mercuric chloride (0.1%) dip for 10 minutes and final treatment with ethyl alcohol (70%) for one minute proved best for culture survival of rhizome tips and rhizome sections at 4 weeks of culture., whereas carbendazim 200 ppm for 15 minutes followed by mercuric chloride 0.1% dip for 3 minutes proved better for culture survival of shoot tip and shoot nodal segments at 4 weeks of culture. In this study protocol for sterilization of different explants leads to the standardization of the explant for tissue culture in Alstroemeria hybrida cv. Pluto.