Campaigners say they will be scrutinising hunts more closely than ever as new legal restrictions come into force on Tuesday.
Provisions from the Hunting with Dogs (Scotland) Act will begin, after MSPs voted for the law earlier this year.
It was designed to close loopholes in the earlier ban on fox hunting, introducing a two-dog limit for all use of dogs in hunting, and also banning trail hunting.
The law also introduces a new licensing scheme, which will allow the use of more than two dogs in certain limited circumstances.
Robbie Marsland, director of the League Against Cruel Sports, said: “Scotland now has the most robust law on hunting with dogs in the UK but this does not mean our job is done, far from it.
“We will be scrutinising the activity of hunts more closely than ever before to see who is complying with the law and who is looking for new loopholes to exploit.
“Throughout the passing of the new legislation there has been a determined and persistent campaign from those desperate to keep the cruelty of hunting alive in our countryside, even now pro-hunters are trying to delay commencement of the law.
“But we are sending a stark warning to hunters that we will be monitoring every hunt in Scotland for as long as it takes until we are satisfied the law is being adhered to.”
Bob Elliot, director of OneKind, said: “Today is a monumental day in Scotland.
“We are thankful to the Scottish Government for listening to the people of Scotland, who have been calling for a real fox hunting ban for more than 20 years, when it became clear that loopholes in the previous law had allowed fox hunting as ‘sport’ to continue.”
The Scottish Greens rural affairs spokesperson, Ariane Burgess MSP, also welcomed the new rules coming into force.
She said: “This is a historic day. We finally have a real hunting ban after decades of campaigning.
“Hunting with dogs is brutal, cruel and outdated. It has no place in a modern or progressive Scotland, and we should all take pride is this new law.
“Loopholes in the original hunting ban were ruthlessly exploited by those who were determined to continue hunting.
“This law is much more robust, but we will be watching carefully to ensure it is effective and abided by.”