In this paper we report the effects of the irradiation of low-density lipoprotein (LDL) by ultra-short laser pulses to obtain in vitro alterations mimicking proatherogenic modifications occurring in vivo in LDL. The modifications by metallic ions (copper and iron) and ultra-short laser pulses were studied by fluorescence steady state and time-resolved lifetime measurements. The results demonstrate that the modifications caused by ultra-short laser pulses and by iron affect the tryptophan residues of apolipoprotein B-100 (Apo-B), slightly decreasing fluorescent lifetimes, with almost no modifications in pre-exponential factors, indicating preservation of structural properties around the fluorophore. On the other hand, oxidation by copper strongly affects the Apo-B protein associated with LDL. We describe a fast, inexpensive, and nondestructive fluorescence-based method that is readily accessible to provide the LDL particle characterization.