Your question is unclear about what you are trying to accomplish. Is it that you want to measure the IC50 for inhibition of an enzyme inside living cells (the term for this is target engagement), as opposed to performing an assay that measures cytotoxicity? What enzyme are you trying to inhibit?

Adam B Shapiro Sir,

I'm trying to inhibit the sPLA2IIA enzyme, which will secreted during the inflammatory condition. I'm inducing the inflammation in normal lung cells and trying to treat the cells with the synthesized drug. Unfortunately, I don't know the treatment dose for the cell lines; hence, how can I determine the drug concentration needed to treat cell lines?

Since the enzyme is secreted, it's the extracellular enzyme that matters, so you don't have to worry about how well the inhibitor penetrates into the cells. Besides the IC50, you also have to worry about the inhibitor becoming bound to medium components such as albumin or lung surfactant, if those are present. This binding will reduce the potency of the inhibitor, because only free inhibitor is capable of inhibiting the enzyme.

The treatment dose should be determined experimentally by varying the inhibitor concentration and measuring the effect at each concentration. You can then relate the effect in the cell culture to the IC50 for enzyme inhibition, if you wish.