You need to calculate the relative activity for each point before entering the data into Prism. Input the normalized activity as an XY table with 3 replicates per X value in subcolumns. You can then run a "Non-linear Regression" with a "Dose-Response - Inhibition" model to find the IC50. You can input the drug concentration either raw or log-transformed, just make sure you select the proper model (i.e. [Inhibitor] or log(Inhibitor))

John Hardy Lockhart , U mean i need to calculate the relative activity of the 3 absorbances value in each concentration? I understand how to run the model, its just the data to choose is making me confused and I didnt get my answer from reading manual. Thank you for your response.

Yes, you'll need to calculate the relative activities for every point in your curve.

% Relative activity = (X - [Average of "no activity" control])/[Average of "normal activity" control]). You can then put these values into Prism to calculate the IC50.

This should give you values ranging from 0 to 1 ± any variance in the controls.

Your "normal activity" control will be your untreated cells. The "no activity" control should be something like a "no substrate" or "no enzyme" (might be good to have both) control.

I see. Thank you very much John Hardy Lockhart