Several systematic errors may occur during the analysis of uninhibited enzyme kinetic data using commercially available multiwell plate reader software. A MATLAB program is developed to remove these systematic errors from the data analysis process for a single substrate-enzyme system conforming to Michaelis-Menten kinetics. Three experimental designs that may be used to validate a new enzyme preparation or assay methodology and to characterize an enzyme-substrate system, while capitalizing on the ability of multiwell plate readers to perform multiple reactions simultaneously, are also proposed. These experimental designs are used to (i) test for enzyme inactivation and the quality of data obtained from an enzyme assay using Selwyn's test, (ii) calculate the limit of detection of the enzyme assay, and (iii) calculate Km and Vm values. If replicates that reflect the overall error in performing a measurement are used, the latter two experiments may be performed with internal estimation of the error structure. The need to correct for the systematic errors discussed and the utility of the proposed experimental designs were confirmed by numerical simulation. The proposed experiments were conducted using recombinant inducible nitric oxide synthase preparations and the oxyhemoglobin assay.