Supplementing dairy diets with rumen-protected lysine (RPL) could help to lower N emissions and improve productive efficiency. The objective of this study was to determine the short-term effects of targeted RPL 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 (mean ± SD) = 687 ± 68 kg; DIM = 151 ± 57] 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 contrasts. Contrast 1 (CONT1): CON compared with NPL and contrast 2 (CONT2): AJP compared with NPL. There were no differences among treatments for either contrast (CONT1 or CONT2) for DMI, BW, DMI as a percent of BW, and BCS (P > 0.05). Milk yield was greater for cows fed NPL (35.98 ± 0.92 kg) than cows fed AJP (34.55 ± 0.93 kg; P = 0.03, CONT2). Milk protein yield was greater for cows fed NPL (1.11 ± 0.03kg) than cows fed AJP (1.06 ± 0.03kg; P = 0.02; CONT2). Milk casein yield was greater for cows fed NPL (0.42 ± 0.02kg) than cows fed AJP (0.34 ± 0.02kg, P = 0.01; CONT2). In conclusion, cows fed NPL performed similarly than cows fed AJP and had increased milk yield and protein yield.