There are three types of eligible participants that need to be included in your number:
Active employees: Start with understanding your plan documents. Your Adoption Agreement and Summary Plan Description detail whether there are any excluded types of participants. Examples of possible exclusions are union employees and leased employees. From there, the document describes the age and service requirements and the entry dates, which will allow you to calculate all eligible active employees.
Former employees: As employees leave the company, they may or may not remove their account balances from the plan. Any employees no longer employed by the company who still have a balance in their account on the first day of the plan year still count as eligible participants.
Deceased employees: Any deceased employees whose benefits have not been fully received by beneficiaries still count as eligible participants.