Swainsonine is a polyhydroxy indolizidine alkaloid with various research and potential therapeutic applications. In this work, swainsonine was partially purified (2.5-folds) with acetone–methanol solvent system from Metarhizium anisopliae fermentation broth. The partially purified broth was further subjected to mass-directed preparative-cum-quantitative analysis. Swainsonine was eluted as MS1 fraction [M + H]+ 174.36 ± 0.21 at 4.91 ± 0.04 min with calculated yield of 7.85 ± 1.59 μg mL−1 corresponding to 3.74 × 105 counts. In situ antiproliferative activity of standard and purified swainsonine fractions was tested against Spodoptera frugiperda, Sf-21 cell line with IC50 values of 2.96 μM and 3.28 μM, respectively, at 36 h. This analytical procedure for purification and quantitative analysis of swainsonine may ensure its suitability for routine laboratory studies and research.