Inactivation of enzymes that may be present in plant tissues is an important first step before isolation of glycosides is attempted. This is especially so when fresh plant material is being processed. Dried material does not require such precaution as these enzymes are active only in fresh tissues.

Due to the wide range of physical and chemical properties of glycosides, it is not possible to follow a single general method for the isolation of all glycosides. Different methods of isolation, based on the nature of the aglycone and the type of sugar–nonsugar linkage of the glycoside are followed for the isolation of specific glycosides.

Following is a general method suited for the isolation of glycosides from plant tissues:

1. The plant part containing the glycoside is carefully and quickly dried and powdered. It is exhaustively extracted with 95% ethanol using hot continuous percolation. Heating deactivates the enzymes. For thermolabile glycosides, the temperature should not exceed 45°C.
2. Alternatively, the fresh plant part is boiled under reflux with 95% ethanol for 30 min for the inactivation of hydrolytic enzymes. The residue of the plant material is then size reduced and further extracted with the same solvent.
3. The combined ethanol extracts or the extract of step 1 is evaporated to dryness in the presence of a little CaCO3 under reduced pressure at a temperature not exceeding 50°C.
4. The dry residue is dissolved in minimum quantity of water (equivalent to 1g/ml of fresh tissue) and 10% basic lead acetate solution is added until there is no further precipitation (of tannins).
5. A further 1/10^th of the earlier added volume of lead acetate solution is added to the solution taken in a mortar.
6. To this mass, add anhydrous sodium sulphate in small portions (1g/ml of lead acetate solution is added first). This is to render the non-glycosidal components insoluble.
7. The above mass thoroughly mixed is allowed to air-dry to become a semi-solid mass.
8. This is then spread out on glass or porcelain tiles and allowed to air dry to a powdery state.
9. The fully dried product is broken up and treated with successive portions of ethyl acetate, ether, chloroform, ethanol, or ethyl acetate-ethanol (4:1). Different solvents are used depending on the solubility of the glycoside.
10. The mixture is filtered and concentrated with ether being added in final stages. The glycoside crystallizes out.
11. Further if the glycosides occur as mixtures, they may be separated using column chromatography, preparative TLC, or fractional crystallization.