The objective of this study was to determine the short-term effects of targeted rumen-protected and post-ruminal Lys supplementation to dairy cows on protein and AA in blood; and production of milk and milk components. A total of 18 multiparous Holstein cows [BW = 687 ± 68 kg; DIM = 151 ± 57 (mean ± SD)] were assigned to 1 of 3 treatments in a replicated 3 × 3 Latin Square design balanced to measure carryover effects. Total length of the experiment was 73 d including a 10 d diet adaptation period before the start of the trial. Experimental periods were 21 d in length with each divided by adaptation phase (d 1 to 14) and sample phase for statistical inferences (d 15 to 20). Treatments were as follows: cows fed a basal TMR + 150g of dried ground molasses (CON); basal TMR+ 150g of dried ground molasses + a commercially available rumen-protected lysine source (AjiPro-L, Ajinomoto Heartland Inc., Tokyo, Japan) (AJP, positive control); and basal TMR + 150g of dried ground molasses + a rumen-protected lysine prototype source (NPL, prototype B, NutraPass 50, ADM Animal Nutrition, Quincy, IL). Protected lysine sources were each included at a rate of 0.51% [w:w] of the DM. Data were analyzed using the MIXED procedure of SAS using 2 treatment orthogonal contrasts. Contrast 1 (CONT1): CON compared with NPL and contrast 2 (CONT2): AJP compared with NPL. Blood was sampled from the coccygeal vein or artery 4 and 8 h after feeding on 3 consecutive days before the first period and at the end of each period from each cow. There were no differences among treatments for either contrasts for total AA concentrations (P > 0.10). Plasma Lys concentration as a percentage of total AA (4.02 vs 4.30 ± 0.09) and plasma Lys concentration as a percentage indispensable AA (8.67 vs 9.30 ± 0.14) was greater for cows fed NPL than cows fed CON (P < 0.01, CONT1). Plasma Lys concentration as a percentage of total AA tended to be greater for cows fed AJP than cows fed NPL (4.43 vs 4.30 ± 0.014;P = 0.08,CONT2). Plasma 3-methyl-histidine concentration tended to be greater for cows fed CON than cows fed NPL (3.362 vs 3.39 ± 0.14 μM/L; P = 0.08, CONT1). In conclusion, Lys lipid protection seems to be an effective method of protection.