If you want to calculate a time difference in Excel, it is important that you understand that Excel stores dates as integers and times as decimal values (see the page on Excel dates for more details). It is only the formatting of an Excel spreadsheet that causes these values to be displayed as dates and times, rather than as simple numbers.
Therefore, when you want to calculate the time difference between two dates and times in Excel, you can simply subtract these values in the same way as you would subtract any other numerical values in Excel. This is shown in the examples below.
When users have problems with Excel date and time difference calculations, these problems are commonly due to incorrect formatting of the results, rather than incorrect calculations. Therefore, in the examples below, we also discuss the cell formatting that you may require, to correctly display the result of your time difference calculation.
Cell B3 of the following spreadsheet shows a simple example of how to calculate a time difference in Excel. The calculation simply subtracts the time in cell B1 from the time in cell B2.
The spreadsheet on the left displays the formula used for the calculation and the spreadsheet on the right shows the result.
Formulas:

Results:

To Format a Cell as a Time:
  Right click on the cell to be formatted 
  Select the option Format Cells... 
  Ensure the Number tab is selected in the window that pops up 
  Select the option Time from the list of Categories and click OK 
In the example above, the time in cell B1 is internally represented by the number, 0.156597222 and the time in cell B2 is internally represented by the number 0.428993056. Subtracting these two numbers gives the result 0.272395833, which, when formatted with the time format, hh:mm:ss, is the time 06:32:15 (i.e. 6 hours 32 minutes and 15 seconds).
The following spreadsheet shows a simple subtraction of two dates and times. Again, the spreadsheet on the left shows the formula used and the spreadsheet on the right shows the result.
Formulas:

Results:

In the example above, the date and time in cell B1 is internally represented by the number, 40562.875 and the date and time in cell B2 is internally represented by the number 40564.625. Subtracting these two numbers gives the result 1.75. I.e. the time difference between the two dates and times is 1.75 days.
Setting Cells to Have the [h]:mm Format:
  Right click on the cell to be formatted 
  Select the option Format Cells... 
  Ensure the Number tab is selected in the window that pops up 
 
Select the option Custom from the list of Categories and type [h]:mm into the box on the right 
  Click OK 
Cell B3 of the above results spreadsheet has the 'general' cell formatting. If you prefer to show the result as a number of hours and minutes, this can be done by setting cell B3 to have the time format [h]:mm, as shown in the spreadsheet below.
A  B  

1  Start Date & Time:  19Jan2011 21:00 
2  End Date & Time:  21Jan2011 15:00 
3  Time Difference:  42:00 
Note that the square brackets in the [h]:mm format definition tell Excel to display the total number of hours, even if this is greater than 24. If the square brackets were not included, Excel would break down the result into a date plus the remaining number of hours.