Haematococcus pluvialis algae can be used to produce the valuable carotenoid pigment astaxanthin. Low value de-proteinized whey permeate (DWP) contains a high concentration of lactose, nitrogen, and ash that algae can use for growth. This project evaluated feeding DWP to H. pluvialis to produce astaxanthin. Astaxanthin produced by H. pluvialis is mainly present in esterified forms, rather than as free-astaxanthin, complicating quantification. A goal of this study was to develop an extraction and de-esterification method to more accurately quantify astaxanthin. Three solvent extraction methods were evaluated for the amount of oil extracted from freeze-dried H. pluvialis biomass. Acetone with grinding in a glass tissue grinder; 2:1 chloroform:methanol with sonication; and 1:1 petroleum ether:diethyl ether with 26% HCl pretreatment; The oil extraction efficiency was 8.9% (SD = 0.97), 14.5% (SD = 0.73), and 33.0% (SD = 0.21), respectively (n = 6). To de-esterify the astaxanthin to allow quantification by HPLC-UV with a free-astaxanthin standard, cholesterol esterase and chemical hydrolysis by KOH were compared. HPLC evaluation determined the cholesterol esterase hydrolysis method was more effective and allowed for quantification of the astaxanthin isomers. Two liters of H. pluvialis stock culture (ca. 25% peak density) were grown in 20 L carboys for 24 h at 4000 lx light under the following 3 feeding conditions, in duplicate: standard algal growth medium; standard algal growth medium + 10 g/L DWP; 10 g/L DWP. Biomass was removed and freeze-dried. Algal oil was extracted using the developed method. Carotenoids were analyzed by HPLC-UV/VIS and MS/MS. Total astaxanthin dry weight was 3.33 μg, 4.40 μg, and 3.61 μg per treatment, respectively. All free-astaxanthin isomers were included in this quantification, pending identity confirmation via MS. These results indicate that H. pluvialis can metabolize DWP, and that DWP alone provides sufficient nutrients for H. pluvialis growth and production of astaxanthin. This is promising for dairy processors, who can utilize this application for DWP as a novel revenue stream.