Secondary school students and teachers are no longer required to wear face masks in classrooms.
The Covid restrictions eased on Monday following an announcement by First Minister Nicola Sturgeon earlier this month.
Despite the change, pupils and staff will still have to wear the coverings when moving around the building and in communal areas such as corridors.
Those who wish to continue to wear a mask will be “fully supported” to do so.
Education secretary Shirley-Anne Somerville hailed the move as a “positive milestone”.
She said: “The last two years have been really tough for our children and young people.
“Teachers and support staff have also had to operate in a challenging environment, but all have worked incredibly hard to deliver a quality learning experience for pupils.
“The removal of the requirement to wear face coverings in classrooms is a positive milestone – it signals the possibility of a brighter future where schools can regain some normality.
“And of course, pupils and staff who continue to choose to wear face coverings in class will be supported in doing so.”
Sturgeon said the relaxation of the rules followed expert advice and was a “further step in allowing children and young people a return to a more normal experience in school after many, many months of sacrifice”.
Restrictions on school assemblies have also been lifted, along with those on transition visits for youngsters starting primary or secondary this August.
In light of the changes, Somerville called for continued vigilance in the fight against the deadly virus.
She said: “We must not forget that Covid-19 has not gone away – we all still need to be on our guard and exercise caution.
“Pupils and staff must still wear face coverings in communal areas and distancing rules still apply.
“The last thing I want is to have measures in place for a minute longer than is necessary, but as we have done throughout this pandemic, we will continue to be guided by scientific and expert advice.”