In this guide, I will explain how to perform an unpaired (independent) T-test in GraphPad Prism. I will also show you how to interpret and report the results.

## Assumptions

Before beginning it is important that your data satisfies the five assumptions of an independent t-test. Refer to our article on this by clicking here.

## The data

To perform an unpaired (independent) T-test in GraphPad Prism you will need to enter two groups of data into separate columns. Upon opening GraphPad Prism, select the ‘**Column**’ type for the ‘**New Table & Graph**’ option. Then select ‘**Enter replicate values, stacked into columns**’ as the ‘**Enter/import data**’ choice.

Alternatively, you can go to ‘**File > New > New Data Table & Graph …**’.

I will use a theoretical example of comparing heights between a group of males and females. So each gender will have a separate column listing all of the height values.

**“There is no difference in height between the males and females”.**

And our alternative hypothesis will be:

**“There is a difference in height between males and females”.**

## Performing the test

To perform an unpaired (independent) T-test, first go to ‘**Insert > New Analysis …**’.

This will open a new window. Here you need to tell GraphPad which test to perform. Select the ‘**t-tests (and nonparametric tests)**’ analysis and make sure the two datasets are ticked on the right window. Click the ‘**OK**’ button.

The next window will ask you to specify whether the t-test is an unpaired (independent) or paired (dependent) test. In this case, select the ‘**unpaired**’ option.

Next, you need to select whether the test will be a parametric or nonparametric test. Under ‘**Assume Gaussian distribution**’, select ‘**Yes. Use parametric test**’. This will ensure an unpaired T-test is performed.

The final option titled ‘**Choose test**’ is referring to whether your two datasets have equal variance. Select, ‘**Unpaired t-test. Assume both populations have the same SD.**’

If you want to change any other settings, such as the confidence level, go to the ‘**Options**’ tab. For our purpose we are going to leave everything in the default settings, but will select ‘**Descriptive statistics for each data set**’ so GraphPad will produce the mean and standard deviation for each group. Click ‘**OK**’ to perform the test.

## Output

The great thing about GraphPad Prism for statistical testing is that the output is very user friendly and self-explanatory. All you need to do is to refer to the ‘**Unpaired t-test**’ section.

- P value – The exact p-value.
- P value summary – A summary of the p-value as represented by asterisks. These are useful to signify the level of significance on graphs, for example.
- Significantly different? (P<0.05) – A straight yes or no answer for the unpaired t-test.
- One- or two-tailed P value? – Whether a one- or two-tailed unpaired t-test was performed.
- t, df – The t statistic and the degrees of freedom in the analysis.

GraphPad also gives you other descriptive information for each dataset (under the ‘**How big is the difference?**’ section) and an output for the F test (under the ‘**F test to compare variances**’ section) to determine if there is an equal variance of data between the two groups.

## Interpretation

By looking at the ‘**Significantly different? (P<0.05)**’ output, a ‘**Yes**’ is given which essentially means that our results are significantly different from each other. The exact p-value is given next to ‘**P-value**’. In this case a p-value of < 0.0001 indicates a very significant difference, since this is less that our level of significance threshold of 0.05.

So, after performing the unpaired T-test in GraphPad we now know that there is a significant difference between the height of males and females in our experiment. In which case, we reject the null hypothesis and accept the alternative hypothesis.

## Reporting

If we were to report the results of this unpaired T-test in a single sentence, we could write:

“On average males (181.1 ± 4.9 cm) were significantly taller than females (163.4 ± 5.1 cm), t(14) = 7.15, p < 0.0001.”

This includes the mean and standard deviation (this information can be found in the ‘**Descriptive statistics**’ sheet) for each group, as well as the t statistic, degrees of freedom and the all-important p-value.

GraphPad Prism version used: 6