The elution characteristics of lovastatin were studied by varying the composition of mobile phase in both isocratic and gradient elution modes to comprehend the role of organic modifier and acidifier on the overall analysis time and retention time of individual forms of lovastatin. Acetonitrile has influenced on the overall analysis time, whereas the acidifier determines the retention time of hydroxy acid form of lovastatin and the retention time gap between the individual forms. A combination of acetonitrile and 0.1% trifluoroacetic acid (TFA) (60:40, v/v) in isocratic elution mode eluted both hydroxy acid and lactone forms of lovastatin at 4.5 and 5.4 min, respectively. This appears to be a better approach for the separation of pharmaceutical and clinical lovastatin samples. At isocratic elution mode, a mixture of acetonitrile and either 0.05% TFA or 0.1% H3PO4 of 60:40 (v/v) has eluted both hydroxy acid and lactone forms of lovastatin at 10 ± 0.5 and 17 ± 0.5 min, respectively. This is suitable for the fermentation-derived samples or for the complex mixtures of structural analogs. The fermentation broth (pH not adjusted) extracted with ethyl acetate at a ratio of 1:1 (v/v) at 60°C for 30 min was the optimal extraction condition for lovastatin.