The line is being piloted for a year and should offer reassurance to staff, Health Secretary Alex Neil said.
Its launch follows the Francis inquiry, which uncovered "appalling" failings at Mid Staffordshire NHS Foundation Trust in England.
Mr Neil said: "It is important that all NHS workers feel that they can raise any concerns they may have safely and confidentially, and following the Francis inquiry, be reassured that health boards will listen.
"All areas of concern must be investigated and lessons will be learned to ensure that the NHS in Scotland is providing the best possible service to patients.
"We already have whistle-blowing policies in place.
"Staff can be assured that these policies will be enforced and the alert line will give staff the best possible support."
NHS staff will be able to use the 0800 008 6112 number, which is being run by Public Concern at Work, an independent organisation.
Any concerns will be passed on to the employer or the relevant regulatory organisation for investigation.
First Minister Alex Salmond announced the plan in November last year.