Preparation of organic thin layers on various special substrates using the pulsed laser deposition (PLD) technique is an important task from the point of view of bioengineering and biosensor technologies. Earlier studies demonstrated that particle ejection starts during the ablating laser pulse resulting in significant shielding effects which can influence the real fluence on the target surface and consequently the efficiency of layer preparation. In this study, we introduce a photoacoustic absorption measurement technique for in-situ characterization of ablated particles during PLD experiments. A KrF excimer laser beam (λ = 248 nm, FWHM = 18 ns) was focused onto pepsin targets in a PLD chamber; the applied laser fluences were 440 and 660 mJ/cm 2 . We determined the wavelength dependence of optical absorption and mass specific absorption coefficient of laser ablation generated pepsin aerosols in the UV-VIS-NIR range. On the basis of our measurements, we calculated the absorbance at the ablating laser wavelength, too. We demonstrated that when the laser ablation generated pepsin aerosols spread through the whole PLD chamber the effect of absorptivity is negligible for the subsequent pulses. However, the interaction of the laser pulse and the just formed particle cloud generated by the same pulse is more significant.B