While her teammates Annemiek van Vleuten and Georgia Williams started the day in the top two positions on general classification, Spratt sensed an early opportunity, escaping from a fractured peloton after the first time up the gruelling nine per cent, six kilometre Uriola climb.
With Spratt soon up the road with a lead of over two minutes, Mitchelton-Scott played the numbers game behind, both Williams and van Vleuten sitting back countering early moves.
At just three seconds behind the Sydneysider on general classification, Italian Elisa Longo-Borghini (Wiggle-High5) tried her luck with 20 kilometres to go, attacking from the chase group with Olga Zabelinskaya (Cogeas-Mettler).
But Spratt was just too strong, holding off the chasing duo to cross the line with over one minute in hand.
"I am really happy," she said. "We came here with some good GC options with Annemiek and Georgia and I was the one people were probably not looking at as much and I used that to my advantage.
"The whole day we were always on the front foot and Annemiek and Georgia could just sit there. I honestly didn't expect to win it but I am super happy. I am really happy for the whole team, it is a great day for us.
"We really used our numbers and isolated Anna van der Breggen (Boels-Dolmans) and Ashleigh Moolman-Pasio (Cervelo-Bigla) and we couldn't have asked for a better day."
Van Vleuten took the race lead after a commanding performance in the ITT on stage 2, with Georgia Williams improving her GC standing following a strong finish on stage 3.
Catch the highlights of the earlier stages here: