Prepare samples with standardized amounts of the substance you are going to assay. Also prepare any unknown samples you want to test. The range of the standardized samples should be large enough to encompass likely measurements from the unknown samples.
Perform the assay on all your samples at the same time. A constant amount of the assay reagent is added to each sample.
Measure the reactions between assay reagents and the samples. Often this is done with a spectrophotometer, which can measure absorption rates of a concentration of substance in a solution.
Create a graph with the amount of the non-reagent substance charted along the x-axis. Use the measurements of your reaction for the y-axis.
Plot data points on the graph for each standardized sample from the assay procedure.
Draw a curve to connect the data points. If the data has a linear relationship, the curve will just be a straight line. This is the standard curve.
Use the graph and measurements from the unknown samples to determine the concentration of each sample. Find the measurement value on the y-axis, follow that point horizontally until you reach the standard curve and then read the x-axis value for that point.