Atheist parents Lee and Lizanne Harris say their children's human rights are being breached because Burford Primary isn't offering any alternatives when they are removed from the assemblies.
They say the children are given an iPad and put in a separate room with a teaching assistant.
Supported by Humanists UK, the couple has been given permission by the High Court to launch a judicial review into the actions of the school.
While the school is run by the Oxford Diocesan Schools Trust, it's not classified as a faith school.
Speaking of why they wanted to take action, the couple said: "We enrolled our children into a state community school - which is meant to have no religious character - but over time we noticed harmful aspects of evangelism spreading into assembly and other parts of the school which goes against our children's rights to receive an education free from religious interference.
"When our children go to school they shouldn't have to participate in Christian prayers, or watch biblical scenes such as the crucifixion being acted out, nor should they have to hear from evangelical preachers who spout harmful and often divisive messages."
Campaign group Humanists UK has long called for an overhaul of the laws surrounding school assemblies.
Currently all schools must have a daily act of broadly Christian worship.
Humanists UK Chief Executive Andrew Copson said: "We are the only sovereign state in the world to require schools to hold daily Christian worship, yet 80% of our young people and 75% of people of parental age are not Christians.
"Our state schools are instead home to children of a broad diversity of cultures and backgrounds and they deserve and need inclusive activities that bring them together as one community.
"Requiring children to participate in religious worship and then marginalising them if in good conscience they cannot, ignores their right to freedom of religion or belief and is a negation of inclusion."
Speaking on Premier's News Hour, Alice Probert from Faith in Schools defending the role of assemblies.
She said: "They help to build community and shared values.
"They encourage moral development, spiritual development and social development so they have a really important place."
Probert did however say those leading assemblies should tread carefully.
"People do need to be trained and equipped so it can be done appropriately so schools and parents are happy," she said. "It's really important we are authentic in our beliefs but to do it in an inclusive way."
In a statement in response to the legal challenge, Anne Davey, chief executive of Oxford Diocesan Schools Trust, said: "ODST is confident that Burford Primary School has acted entirely appropriately, and has followed statute in ways that are similar to all local or indeed national schools.
"It has provided exactly what the law requires, which includes provision for children to be withdrawn if parents so request."
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