AMs will need receipts for all their expenses claims from today.
They will also lose the right to buy furniture and fittings for their second homes at the taxpayers' expense.
The new rules are among recommendations made by the Jones Review into expenses, published after a 10-month inquiry and accepted in full by the Assembly's authorities in July.
All 108 changes are due to be in place after the next election in 2011.
The first 28 come into force today, scrapping a flat-rate allowance of £30.65 available to AMs when they are away from their main home on business.
All expenses claims will have to be accompanied by "documentary evidence’’ and an explanation of why they were incurred.
Eligible AMs can still be reimbursed for utility bills, council tax, broadband, insurance and a TV licence for their second homes in Cardiff.
Presiding Officer Lord Elis-Thomas also wants AMs to give up control of their salaries.
He is overseeing legislation that would create an independent board to set AMs' pay and allowances.
It would also cut a link with wages for MPs. AMs currently earn 82% of an MP's salary.
Conservative AMs have said they will not support the proposed new salary arrangements, arguing that the shake-up could drain resources.
Lord Elis-Thomas said the all-party Assembly Commission which handles day-to-day business in Cardiff Bay wanted to "demonstrate to the Welsh public a commitment to transparency’’.
"A commitment that was made well before the storm over MPs expenses blew up in Westminster,’’ he added.
"The panel's recommendations offer a unique opportunity to strengthen the National Assembly's contract with the people of Wales in its role of law-making and government scrutiny.
"We have been able to take a lead on this issue, so distancing ourselves from the turmoil in Westminster, thanks in large part to the swift and supportive response that Assembly Members gave to my statement to them back in July.’’
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