Welfare reforms mean many will lose as much as £35 a week as part of a change from Disability Living Allowance (DLA) to Personal Independence Payments (PIP).
Under the new system anyone who can walk more than 20 metres is no longer entitled to the £56.75 enhanced weekly mobility allowance and could be offered the standard rate of just £21.55.
A distance of 50 metres has been used as a measure of significant mobility impairment by successive governments for the past 35 years.
It has been estimated that as many as 80,000 working age disabled people in Scotland will lose either some or all of the mobility allowance as a result of the changes.
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Today's legal challenge, by a group of 50 charities known as the Disability Benefits Consortium could force the UK Government to rethink the reform.
They claim that the official consultation process did not mention the new limit would be reduced to only 20 metres.
The PIP 20 metre rule Judicial Review case is due to be heard at the High Court in Birmingham later.
Claire Nurden, co-chairman of the Disability Benefits Consortium, said: "PIP is intended to help those most in need but it is exactly these people that are set to lose. The 20 metre rule is arbitrary; it's inconsistent with guidance used for the past 35 years which suggests that 50 metres is a more appropriate measure of significant mobility impairment.
"The Government has presented no new evidence to justify the change.
"This money is an absolute lifeline for people - many use it for a Motability vehicle so they can continue to remain independent, travelling to work or medical appoint-ments, or picking up the children from school.
"We have been over-whelmed by the response we have had from disabled people terrified about what this rule will mean for them.
"We hope the court case finds the decision unlawful. It is vital that this prompts the Government to reinstate the 50 metre distance."
The Disability Benefits Consortium also claims the Government has failed to justify the decision to change the distance.
Ms Nurden added: "The Government has argued that the change is justified because the test considers whether people can complete the distance 'reliably', but this fails to address the issue because those who can walk between 20 and 50 metres will still lose out."
A spokeswoman for the Department for Work and Pensions said: "We believe the current assessment criteria, including the 20 metres distance, are the best way of identifying those whose physical mobility is most limited."