Williams, who won the Madrid Open in 2012 and 2013, is due to be replaced by her compatriot Coco Vandeweghe in the tournament starting on Saturday, reports Efe.
"We regret Serena's absence and we wish her a speedy recovery," tournament director Manolo Santana said.
"She is one of the greatest champions of the sport, she has always given good performance in the tournament and we hope to see her in the future," Santana added.
Williams is expected to return to competition at the Italian Open in Rome, set to occur between May 14 and 22.
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