Are you measuring the decrease in fluorescence of NADH (excitation wavelength 340 nm, emission wavelength 460 nm)? Can you do a continuous measurement, meaning measuring the fluorescence of each sample multiple times at evenly-spaced time points? This is useful because it allows you to measure the initial rate of reaction as the slope of the progress curve, instead of taking just a single measurement. It is ESSENTIAL for the kinetic analysis that all rate measurements are initial rates. Measuring slopes allows you to be sure of this.

Choosing a suitable enzyme concentration is crucial to make sure you have enough time to measure the initial rate, and enough of a signal when you do.

Make sure the volume you are dispensing with the electronic pipettor is within the designed range for that instrument. If you try to dispense too small a volume, the results may have a lot of variability. Make sure you are not introducing any bubbles into the samples by expelling air from the tips.

If you are making the fluorescence measurements in a multiwell plate using a plate reader, you should be using a solid black plate and reading from the top with no cover. Your reaction buffer should contain a non-ionic detergent to keep things from sticking to the wells. I prefer 0.01% Triton X-100. This also helps with mixing by reducing the surface tension of the solution, and assures that all wells have the same meniscus shape.

Be sure to thoroughly mix the samples. This is easy to do in a 96-well plate, but a little trickier with a 384-well plate.

Plate readers usually allow you to choose some of the settings for the measurements, including the number of flashes and the gain. Increasing the number of flashes improves the results, up to a point. Be sure to adjust the gain to an appropriate level to avoid exceeding the linear dynamic range of the detector. Some plate readers have a focus adjustment, too, which only needs to be done one time as long as the volume stays the same. (Needless to say, all wells must have the same volume.)

Choosing a suitable range of substrate and inhibitor concentrations is essential for obtaining a high quality kinetic MOI analysis. Substrate concentrations should range from below to above their Kms, such as 1/5 to 5 times the Km. The inhibitor concentration should be chosen to cover the dynamic range of effect, both above and below the IC50. It may take a few tries to get this right.

As for the data analysis, a modern approach is to use nonlinear regression to fit the matrix of initial rate measurements made while varying substrate A, substrate B and inhibitor concentrations to the kinetic rate equation for the Ping Pong mechanism modified to include competitive inhibition with respect to either substrate A or substrate B. The older approach involves various reciprocal plots and replots. See the textbook by I. Segel (https://www.amazon.com/Enzyme-Kinetics-Behavior-Equilibrium-Steady-State/dp/0471303097) for kinetic rate equations and plot-based analysis of kinetic data.