Guus Hiddink's side face Mexico in a friendly in Amsterdam on Wednesday, before taking on Latvia at the same venue in a Euro 2016 Group A qualifier on Sunday.
But Milan midfielder De Jong will not be present, with the Dutch Football Association confirming the news on Monday.
"Nigel de Jong signs off with hamstring injury," a brief statement on their official Twitter account read. "Hiddink will decide later today about a possible replacement."
The announcement comes shortly after the news that Ajax midfielder Davy Klaassen had pulled out with a groin problem, to be replace by QPR's Leroy Fer.
After finishing third at the World Cup in Brazil, Netherlands have made a stuttering start to their Euro 2016 qualifying campaign, with just three points to their name from the opening three matches following surprise defeats to Czech Republic and Iceland.
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