We investigate photocycle excitation of crystalline photoactive yellow protein using femtosecond laser pulses. This work establishes the feasibility and suitable optical excitation conditions to perform femtosecond time resolved X-ray crystallographic measurements using an X-ray free electron laser. Flash photolysis experiments demonstrated photocycle yields of the long-lived ‘pB’ signalling state of PYP of up to 10% with pulse durations of 130, 500 and 850 fs at 450 nm wavelength. The power density dependence of the transient pB concentration depends strongly on the pulse duration primarily because photobleaching is prominent at the GW/mm2 level.